Humanized CC chemokine receptor 4 (CCR4) antibodies and methods of use thereof

ABSTRACT

The present invention provides humanized monoclonal antibodies, bi-specific antibodies, antibody conjugates, and fusion proteins that bind to the chemokine receptor CCR4. This antibody is derived from CCR4-IgG1 and recognizes the same epitope. This antibody contains either an IgG4 or a stabilized IgG4 in order to improve binding efficiency and reduce in vivo Fab arm exchange. Binding of the antibodies disclosed herein to CCR4 inhibits ligand-mediated activities and is used to treat symptoms of cancer.

RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 15/517,108, filed on Apr. 5, 2017 which is a national stage entry ofPCT Application No. PCT/US2015/054202, filed on Oct. 6, 2015, whichclaims priority to, and the benefit of U.S. Provisional Application No.62/060,381 filed on Oct. 6, 2014, the contents of which are incorporatedby reference in its entirety.

GOVERNMENT INTEREST

This invention was made with government support under Grant No. CA093683awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

This invention relates generally to the anti-CCR4 monoclonal antibodieshaving an IgG4 Fc domain, as well as to methods for use thereof.

INCORPORATION-BY-REFERENCE OF SEQUENCE LISTING

The contents of the text file named “5031461-034US3 SL.txt”, which wascreated on Sep. 28, 2020 and is 35,098 bytes in size, are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Cutaneous T cell lymphoma (CTCLs) is the second most common extranodalnon-Hodgkin's T cell lymphomas in adults. A recent WHO-EORTC consensusclassification (Willemze R. et al. Blood 2005, 105:3768-3785) indicatesthat there are thirteen clinically and histologically distinct types ofCTCL; however, 90% of CTCLs fall into three classes; mycosis fungoides(MF), primary cutaneous anaplastic large cell lymphoma (ALCL), andSezary syndrome. The most common type of CTCL, mycosis fungoides, ischaracterized by erythematous patches and plaques that most commonlycontain CD4⁺ T cells that show an affinity for the epidermis, orepidermotropism (Willemze R. et al. Blood 2005, 105:3768-3785). Stagingis based upon a TNM classification; patients with Stage 1A disease havenormal life expectancies, while patients with Stage 1B or greater have adiminished life expectancy (Kim, Y. H. et al. Arch Dermatol 2003,139:857-866). Patients with Stage II-IV disease have a median survivalof less than five years, with large cell transformation often leading toaccelerated deterioration (Kim, Y. H. et al. Arch Dermatol 2003,139:857-866). Sezary syndrome is a leukemic variant of CTCL whereinclonal CD4⁺ T cells accumulate in blood and lymph nodes as well as skin;five year survival is less than 25%. Primary cutaneous ALCL has a muchless aggressive course, with a five year survival of 95%; however,cutaneous ALCL with concurrent nodal involvement is more aggressive(Willemze R. et al. Blood 2005, 105:3768-3785; Kadin M E, Carpenter C.Semin Hematol 2003, 40:244-256).

There is significant immune dysfunction in CTCL patients, with globaldysregulation of the T cell repertoire of unknown etiology (Yamanaka K.et al. Clin Cancer Res 2005, 11:5748-5755; Yawalkar N. et al. Blood2003, 102:4059-4066). The terminal event in most patients is bacterialsepsis. Current therapies for advanced MF and Sezary syndrome arepalliative and durable long-term remissions are rare (Querfeld C. et al.Curr Opin Hematol 2005, 12:273-278). Thus, there is an urgent need formore effective therapies.

Current hypotheses indicate that aberrant T-cell activity is a likelydriver in the pathophysiology of CTCL. Of particular importance is theobservation that malignant T-cells may play a role akin to regulatory Tcells and as such supress antitumor activity in CTCL. The CC chemokinereceptor 4 (CCR4) is expressed at high levels on malignant, skin homingT-cells that are present in CTCL, as well as on regulatory T-cells(Tregs). Tumor cells secrete the chemokines CCL17 and CCL22 which areligands for CCR4. In turn, secretion of these ligands attractsregulatory T-cells and malignant T-cells to the sites of the tumor,which results in a suppression of effector T-cells in the cancer cellenvironment. This suppression of the effector T-cells results in afavourable environment for continued cancer cell growth.

Previous work indicated that the use of a humanized anti-CCR4 monoclonalantibody, mAb2-3 IgG1, reduced the migration of regulatory T cellstoward the CCR4 ligands, and ultimately resulted in a decrease of tumorsize in in vivo tumor models. Regulation of both the migratory abilityof regulatory and malignant T-cells toward the tumor cells, as well asdirect cellular toxicity of the malignant cells, plays a key role in theprogression of the cancer.

SUMMARY OF THE INVENTION

The invention provides an isolated humanized monoclonal antibody thatbinds to the human CC chemokine receptor 4 (CCR4) and has an IgG4 heavychain constant region

The invention further provides an antibody containing a V_(H) amino acidsequence having SEQ ID NO: 2 and a V_(L) amino acid sequence having SEQID NO: 4; a V_(H) amino acid sequence having SEQ ID NO: 16 and a V_(L)amino acid sequence having SEQ ID NO: 18; a V_(H) amino acid sequencehaving SEQ ID NO: 20 and a V_(L) amino acid sequence having SEQ ID NO:22; a V_(H) amino acid sequence having SEQ ID NO: 24 and a V_(L) aminoacid sequence having SEQ ID NO: 26; a V_(H) amino acid sequence havingSEQ ID NO: 28 and a V_(L) amino acid sequence having SEQ ID NO: 30; or aV_(H) amino acid sequence having SEQ ID NO: 44 and a V_(L) amino acidsequence having SEQ ID NO: 46 and a heavy chain constant region havingSEQ ID NO: 6 or SEQ ID: 8.

The antibodies according to the invention have a binding affinity ofabout 1.5 nM⁻¹ or less.

The invention also provides a antibody that is a bi-specific antibodycontaining the antibody according to the invention and the heavy-lightchain of an antibody that recognizes a second antigen. For example, thesecond antigen is a tumor associated antigen or a T-cell functionmodulating molecule. The tumor associated antigen is for example CA-IX,ErbB2 or HVEM. The T-cell function modulating molecule is PD-L1, GITR,IL21, IL21R, CD160, TIM3, LAG3 or GALS

In another aspect, the invention provides a cell producing theantibodies of the invention.

In a further aspect, the antibody is linked to a therapeutic agent. Thetherapeutic agent is, for example a toxin, a radiolabel, a siRNA, asmall molecule, or a cytokine. The cytokine is, for example, IL-2 orTGF-beta.

The invention further provides fusion proteins containing the antibodiesof the invention. A fusion protein is, for example, an anti-CCR4antibody or a functional fragment thereof, operably linked to a cytokineor growth factor, such as an IL-2 or TGF-beta polypeptide.

The invention further provides methods for increasing T cellproliferation by contacting a T cell with a fusion protein containing ananti-CCR4 antibody operably linked to a cytokine.

In some aspects the invention provides a method of inhibiting themigration of regulatory T-cells (Tregs) in a subject by administering tothe subject an antibody according to the invention. The lymphocytes,effector T-cells or Tregs are not depleted.

Also included in the invention is a method for augmenting an immuneresponse to an antigen by contacting the antigen an antibody accordingto the invention. In a further aspect, the antibody is administeredprior to or after exposure to the antigen. The administration of theantibody of the present invention causes an increase in antigen-specificT-cell activity. In another aspect, the administration of the antibodyof the present invention causes an increase in T-cell proliferation. Forexample, the T cell is an effector T-cell. For example, the antigen is aviral antigen, a bacterial antigen, or a tumor associated antigen. Inone aspect, the viral antigen is, for example, HIV.

The invention also provides a method for reversing regulatory Tcell-mediated suppression of effector T cell proliferation comprisingcontacting a T cell with an antibody according to the invention.

In another aspect, the invention provides a method for treating oralleviating a symptom of cancer by administering to a subject in needthereof a composition including an antibody according to the invention.The cancer is, for example, a solid cancer or a hematologic cancer.Exemplary hematologic cancers include, but are not limited to: cutaneousT-cell lymphoma (CTCL), mycosis fungoides (MF), primary cutaneousanaplastic large cell lymphoma (cutaneous ALCL), Sezary syndrome, oradult T cell Leukemia/Lymphoma (ATLL). Exemplary solid cancers include,but are not limited to: renal cell carcinoma, breast cancer, lungcancer, ovarian cancer, prostate cancer, colon cancer, cervical cancer,brain cancer, liver cancer, pancreatic cancer, kidney or stomach cancer.The cancer is a solid cancer or a cancer that overexpresses CA IX,PD-L1, or HVEM.

The administration routes, in any methods of this disclosure, include,but are not limited to parenteral, (e.g., intravenous), intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration.

The subject in any methods of this disclosure is, for example, a mammal.The mammal is, for example, a human.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the present invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are expressly incorporated byreference in their entirety. In cases of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples described herein are illustrative onlyand are not intended to be limiting.

Other features and advantages of the invention will be apparent from andencompassed by the following detailed description and claims

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 . mAb2-3 mediates inhibition of Treg migration. (A) The CCL22expression on ovarian cancer cell lines is shown. Cancer cells weretreated with or without brefeldin-A. After a 4-hour treatment, cellswere harvested, stained with anti-CCR4 antibody, and then analyzed byflow cytometry. (B) In vitro chemotaxis of CD4+CD25+ T cells induced byCCL22-expressing ovarian cancer cell supernatant was inhibited bymAb2-3, but not by the control antibody. (C) Chemoattraction of humanlymphocytes by 100 nM CCL22 is inhibited by mAb2-3 in a dose dependentmanner. Results were expressed as means±SD and Student's t-test. Thesedata indicate that ovarian cancer cell lines, IGROV-1 and OVCAR-5, arecapable of CCL22 secretion and may significantly contribute to theincreased CCL22 chemoattraction on Tregs. In addition, both mAb2-3 IgG1and IgG4 are able to inhibit the CCL22 chemoattraction on Tregs.

FIG. 2 . In vivo Chemoattraction of human lymphocytes by CCL22-secretingIGROV-1 xenograft is inhibited by mAb2-3. (A) Mice bearing ovarian tumorcells were injected with luciferized CD4+CD25+CD127dim/− Treg cells andtreated with of 3 mg/kg of mAb2-3 IgG1 or IgG4 or an equal volume ofPBS. The in vivo bioluminescence images of the ovarian cancer xenograftmouse model at 18 hours (imaging) post-injection of luciferizedCD4+CD25+CD127dim/− T cells. (B) The bioluminescence intensity of tumorregion was measured by IVIS imaging system and software. (C) Tumors wereharvested and treated with collagenase, stained with anti-CD4 andanti-CD25 antibodies, and then analyzed by flow cytometry. Thepercentage of CD4+CD25+ Tregs is shown. Statistical values were computedwith Student's t-test. * and **, represent p value<0.5 and 0.01,respectively.

FIG. 3 . Validation of human PBMCs distribution in vivo. The FACS plotsand graphs in (A) and (B) depict the measurement of the CD4+ T cellpopulation after in vivo treatment with anti-CCR4 antibodies. First,mice were received 1×10⁷ human PBMCs through tail vein and then 3 mg/kgof antibodies were injected intravenously into mice. After a 24 hourcirculation period in vivo, mouse blood was collected and human PBMCswere stained with Pacific blue conjugated anti-CD3, Brilliant Violetconjugated anti-CD4, APC conjugated anti-CD25, PE-Cy7 conjugatedanti-CD127, PE-Cy5 conjugated anti-CD45RA, and PerCp-Cy5 conjugatedanti-CCR7 antibodies, and gated to distinguish Tregs, Tcms, Tems, Teffs,and Tnaives. (C) The percentage of CD25+CD127− Treg, (D) CD45RA+CCR7−Teff, (E) CD45RA+CCR7+ Tnaive, (F) CD45RA−CCR7− Tem, and (G)CD45RA−CCR7+ Tem population in CD3+CD4+ T cells are shown from theaverage of three independent experiments. *, p value <0.05. (H)Quantification of in vivo T cell population after antibody treatment. *,p value <0.05. In this experiment, mAb2-3 IgG1 mediated Treg depletionand decreased naïve T cell population. In the contrast, the IgG4 formatof mAb2-3 lost the depletion activity on T cells and maintains thenormal T cell subpopulations in vivo.

FIG. 4 . Generation and verification of tumor-primed T cells to IGROV-1ovarian cancer cells. (A) The verification of monocyte-derived dendriticcells (DCs). (B) Human tumor-primed T cells were incubated withtumor-pulsed DCs or unpulsed DCs. After a 48-hour incubation, thesupernatant was harvested and IFN-gamma was measured by MSD. (C) Humantumor-primed T cells were incubated with IGROV-1 or Tregs. Thesupernatant was harvested, IFN-gamma was measured by MSD, (D) and theLDH was measured by ELISA to detect the cell death percentage. Resultsare expressed as mean±S.D. and Student's t-test.

FIG. 5 . Immunomodulatory therapy of mAb2-3 in IGROV-1 xenograft micebaring IGROV-1-primed T cells. (A) Mice were inoculated with 5×10⁶IGROV-1 tumor cells. When the tumor reached the size of 50 mm³, 1×10⁷IGROV-1-primed T cells, 1×10⁶ Tregs and 1 mg/kg of antibodies wereinjected through tail veil. Mice were treated twice per week. Tumorsizes were calculated by caliper and measured as length×(width) 2×0.52.Grey *, p<0.05, compared to mAb2-3 IgG1; Black * and **, p<0.05 andp<0.01, compared to mAb2-3 IgG4. (B) Tumors were harvested and weighted.Bar, 1 cm. (C) Mice body weight were monitored through the entireexperiment. (D) After antibody treatment, the T cell subpopulations inhuman T cells were measured by flow cytometry. In these NSG mice bearingtumor-primed T cells showed enhanced killing activity after treatingwith mAb2-3 IgG1 or IgG4. These results show that both Treg depletionand inhibition of Treg recruitment by mAb2-3 are beneficial to tumortreatment.

FIG. 6 . Signaling inhibited by the humanized anti-CCR4 antibody. (A)Human PBMCs were incubated with or without Tregs in the presence andabsence of control antibody, mAb2-3 IgG1, or mAb2-3 IgG4. PBMCsproliferation was detected by [3H] thymidine incorporation (CPM). (B)CD4+ T cells were isolated from human PBMCs and further incubated withTregs in the presence and absence of control antibody, mAb2-3 IgG1, ormAb2-3 IgG4. Senescence-associated beta-galactosidase (SA-β-gal)positive cells were quantitated. (C) Western blotting conducted withanti-p-ERK1/2, ERK1/2, p-P38, and P38 antibodies and Western blotanalyses; folds of p-ERK1/2 and p-P38 expression compared with thecontrol IgG treated CD4+ T cells were determined. (D) The expression ofCD27 and CD28 on CD4+ T cells were detected by flow cytometry aftertreated with control IgG, mAb2-3 IgG1, or mAb2-3 IgG4. All values aremean±S.D. These data revealed the additional function of mAb2-3 toinhibit Treg activity by reducing the expression of SA-β-gal and p-P38and restoring the expression of CD27 and CD28, which provideco-stimulatory signals and are required for lymphocyte activation.

DETAILED DESCRIPTION

Chemokines are a family of secreted proteins known primarily for theirroles in leukocyte activation and chemotaxis. Their specific interactionwith chemokine receptors on target cells trigger signaling cascades thatresult in inflammatory mediator release, changes in cell shape, andcellular migration. The CC chemokine receptor 4 (CCR4) is the cognatereceptor for the CC chemokines CCL17 and CCL22, and is expressed onfunctionally distinct subsets of T cells, including T helper type 2cells (Th2), and the majority of regulatory T cells (Tregs) (Iellem etal., 2001; and Imai et al., 1999). Growing evidence indicate thatCCL17/22 secretion promotes increased numbers of tumor-infiltratingTregs by malignant entities such as colorectal, ovarian, Hodgkin'slymphoma and glioblastoma (Curiel et al., 2004; Wagsater et al., 2008;Niens et al., 2008; Jacobs et al., 2010; Hiraoka et al., 2006).Increased levels of Treg in tumors hinder efficient antitumor immuneresponses (Wood et al., 2003; and Levings et al., 2001) and are oftenassociated with poor clinical outcome and tumor progression (Hiraoka etal., 2006; and Woo et al., 2001). Accordingly, one major obstacle ofsuccessful cancer therapies might be caused by migration of Treg intotumors and their suppression of antitumor immune responses in the tumormicroenvironment (Zou et al, 2006; and Yu et al, 2005). In an effort toabrogate Treg suppressive function and consequently promote antitumorimmunity, monoclonal antibodies (mAbs) as immunotherapeutics againstTregs have been evaluated in preclinical and clinical studies in recentyears (Mahnke et al., 2007; Roncarolo et al., 2007). However, a caveatto systemic Treg depletion with mAb immunotherapy is its highlyanticipated association with autoimmunity (Sakaguchi et al., 2008; andKohm et al., 2006). An alternative strategy to avoid Treg induced cancerimmune evasion is to develop a tumor-associated Treg targeting therapythat directly hinders Treg attraction and accumulation in tumor tissue.

One potential of mAbs in cancer immunotherapy lies in their capacity toblock or modulate immunological axes which promote immune evasion bytumors. The chemokine receptor CCR4 is highly expressed on the majorityof FOXP3⁺ Tregs, immune cells which are considered the most potentinhibitors of anti-tumor immunity and the greatest barrier to successfulimmunotherapy (Baatar et al., 2007). Moreover, the tumor-associatedchemokines of CCR4 have been detected in patients with different typesof cancer (Mizukami et al., 2008; Gobert et al., 2009; and Faget et al.,2011). Thus, the targeted approach of human anti-CCR4 mAb immunotherapydescribed herein offers significant advantages in improving cancerimmunotherapeutic efficacy while simultaneously reducing its sideeffects.

The present invention provides humanized IgG4 monoclonal antibodiesspecific against chemokine (C-C motif) receptor 4 (CCR4). The initialhumanization of the anti-CCR4 antibodies is described in WO 2009/086514the contents of which are incorporated by reference in its entirety. Thedescription of an optimized variant of the humanized anti-CCR antibody,Ab2-3 IgG1, is described in WO 2013/166500 the contents of which areincorporated by reference in its entirety. The antibodies were producedby humanizing a mouse anti-CCR4 monoclonal antibody, mAb1567 thatrecognizes the N-terminal and extracellular domains of CCR4. Unlikeaffinity maturation of antibodies against antigens for which pureprotein is readily available, affinity maturation of anti-CCR4antibodies was particular challenging due to 7-transmembrane structureof the protein. This complex structure of CCR4 made screening andselection affinity matured antibodies less efficient and lesspredictable.

A humanized, IgG4 isotype monoclonal antibody against CCR is describedherein and henceforth referred to as CCR4-IgG4. CCR4 IgG4 has adifferent Fc domain amino acid sequence in comparison to CCR4-3 IgG1.The CCR4-3 IgG4 antibodies have affinities that are at least equal to or1-fold, 1.5-fold, 2-fold higher than the originally described CCR4-IgG1.

The CCR4-IgG4 antibodies of the invention also effectively inhibit thechemotaxis of CD4⁺CD25^(high) Tregs. The CCR4-IgG4 antibodies of theinvention also mediate the activation of tumor-primed T-cells byinhibition of the Treg recruitment from the tumor tissue. ImportantlyCCR4-IgG4 antibodies are non-immunodepleting.

Accordingly, the CCR4-IgG4 antibodies are useful in treatingCCR4-expressing tumors such as cutaneous T-cell lymphoma. Additionally,the affinity optimized anti-CCR4 antibodies are also useful in thetreatment of other tumors by enhancing the anti-tumor immune response,by suppressing Treg trafficking.

Cutaneous T-cell lymphomas (CTCLs) are a heterogenous group oflymphoproliferative disorders causes by clonally derived skin homing Tcells. CTCL cells uniformly express CCR4. Specifically, CCR4 is aprominent feature of malignant T cells in MF, cutaneous ALCL, androughly 50% of nodal ALCL. Unlike CLA, it is reliably expressed inSezary syndrome and during large cell transformation of MF and is alsoexpressed by other T lymphoid malignancies that can involve skin, suchas Adult T Cell Leukemia/Lymphoma (ATLL). Expression of CCR4 is limitedamongst non-malignant cells and absent on neutrophils, monocytes, or Bcells. Importantly, CCR4 is absent on naïve T cells, and present onfewer than half of all memory T cells. The reliable expression of CCR4on CTCL cells, and its limited expression on other immune cells, makestargeted therapy of CCR4 an attractive goal for these malignancies.

While some progress has been made in identifying small moleculeinhibitors that are relatively selective for CCR4, specific monoclonalantibodies against CCR4 are an attractive target for immunotherapy ofCTCL because of their exquisite binding specificity. In addition, the invivo effector functions that are mediated through Fc binding to Fcγreceptors can be exploited to kill tumor cells. The precise propertiesof Mabs that are required for optimal in vivo immunodepleting activityare not known, but antibodies can be selected to act as either asreceptor agonists or antagonists, and/or to promote or inhibit receptordimerization and/or internalization. Different immune mechanisms ofantibody-mediated tumor clearance have also been identified. Forexample, Mab-mediated recruitment of natural killer cells to tumors canoccur through the Fc-γ activation of receptors on these immune effectorcells, a process known as antibody-dependent cellular cytoxicity (ADCC).Other immune mechanisms include complement dependent cytotoxcicity (CDC)and antibody dependent cellular phagocytosis (ADCP). Additionalmechanisms related to intrinsic Mab activities include: blockade ofligand binding or hetero-dimerization, inhibition of downstreamsignaling of Akt, and acceleration of receptor internalization. Thelatter mechanism is particularly effective because ligand-inducedendocytosis and degradation of active receptor tyrosine kinases (RTKs)is considered a major physiological process underlying attenuation ofgrowth-promoting signals.

Leukocyte trafficking, which is critically regulated by chemokines andtheir receptors, share many of the characteristics of tumor cellinfiltration and metastasis. While expression of the chemokine receptorCCR4 by tumor cells is associated with skin involvement, CCR4 also hasan important role in both normal and tumor immunity. In a subset of CTCLpatients with HTLV-1 associated Adult T-cell leukemia/lymphoma (ATLL),the tumor cells themselves function as regulatory T (Treg) cells,contributing to tumor survival in the face of host anti-tumor immuneresponses. In other types of cancers, the chemokines TARC/CCL17 andMDC/CCL22, specific ligands for CCR4 that are produced by tumor cellsand the tumor microenvironment, attract CCR4⁺ Treg cells to the tumor,where they create a favorable environment for tumor escape from hostimmune responses. Thus, a therapeutic anti-CCR4 Mab is the idealtreatment modality for many different cancers, not only to directly killthe CCR4⁺ tumor cells, but also to overcome the suppressive effect ofCCR4 Treg cells on the host immune response to tumor cells.

In one aspect the present invention provides a high affinity humanizedmonoclonal antibody that specifically binds CCR4 proteins that modulatesT-cell recruitment without inducing lymphocyte depletion. Binding ofthis antibody to the CCR4 receptor, interrupts ligand or agonist bindingof CCR4. Exemplary ligands or agonists that compete for binding to theCCR4, and which are blocked in the presence of the invented antibody,include, but are not limited to, CCL17, CCL22, and vMIP-III. By avariety of mechanisms, the antibody may decrease ligand-inducedchemotaxis of CCR4-expressing cells, such as cutaneous T cell lymphomacells (CTCL cells) or ovarian cancer cells. The CCR4-IgG4 antibody ismonovalent or bivalent and comprises a single or double chain. TheCCR4-IgG4 antibody may also be a bi-specific antibody, wherein at leastone of the heavy-light chain heterodimers recognizes CCR4. Functionally,the binding affinity of the CCR4-IgG4 antibody is about 1.5 nM⁻¹ orless. The glycosylation of the Fc region of the antibody is modified toalter CCR4 binding or CCR4 ligand-blocking characteristics. Forinstance, the fucose content of the Fc region is decreased compared towild type. Furthermore, the antibody comprises a therapeutic agentincluding, but not limited to, a toxin, a radiolabel, a siRNA, or acytokine.

The CCR4-IgG4 modulates T cell migration and activity. Specifically, theCCR4-IgG4 can block, inhibit or decrease the migration of Tregs towardCCL ligands and as a result reduce the suppressor activity of Tregs, forexample, regulatory T cell-mediated suppression of T cell activity. Inanother aspect, the CCR4-IgG4 can augment an immune response to anantigen. For example, the CCR4-IgG4 increases antigen-specific T cellactivity. In other aspects, the CCR4-IgG4 restores or increases T cellproliferation, for example, effector T cell proliferation. In a furtheraspect, the CCR4-IgG4 activates T cells to secrete cytokines, such asIFN-γ. The CCR4-IgG4 also has a potent immunomodulation effect onregulatory T cells and effector T cells, resulting in inhibition of Tregrecruitment to the tumor tissue and consequent activation of effector Tcells against the tumor cells.

Unlike the previously described CCR4-IgG1, CCR4-IgG4 described hereindoes not induce cell death by complement-dependent cytotoxicity (CDC),antibody-dependent cellular toxicity (ADCC), antibody dependent cellularphagocytosis (ADPC), or any other known mechanism.

The nucleic acid and amino acid sequence of the engineered CCR4-IgG4antibody is provided below in Tables 1A-2B.

Another aspect of the current invention, includes a stabilized versionof the CCR4-IgG4 monoclonal antibody, wherein amino acid substitutionsin the hinge region allows for antibodies that do not undergo in vivoFab arm exchange, resulting in more efficient antibody binding.

TABLE 1A  mAb2-3 IgG4 Variable Region nucleic acid sequencesV_(H) chain of mAb2-3 IgG4 (SEQ ID NO: 1)CAGGTGCAGCTGGTGCAGAGCGGCGCGGAAGTGAAAAAACCGGGCGCGAGCGTGAAAGTGAGCTGCAAAGCGAGCGGCTATACCTTTGCGAGCGCGTGGATGCATTGGATGCGCCAGGCGCCGGGCCAGGGCCTGGAATGGATTGGCTGGATTAACCCGGGCAACGTGAACACCAAATATAACGAAAAATTTAAAGGCCGCGCGACCCTGACCGTGGATACCAGCACCAACACCGCGTATATGGAACTGAGCAGCCTGCGCAGCGAAGATACCGCGGTGTATTATTGCGCGCGCAGCACCTATTATCGCCCGCTGGATTATTGGGGCCAGGGCACCCTGGTGACCGTGAG CAGCV_(L) chain of mAb2-3 IgG4 (SEQ ID NO: 3)GATATTGTGATGACCCAGAGCCCGGATAGCCTGGCGGTGAGCCTGGGCGAACGCGCGACCATTAACTGCAAAAGCAGCCAGAGCATTCTGTATAGCAGCAACCAGAAAAACTATCTGGCGTGGTATCAGCAGAAACCGGGCCAGAGCCCGAAACTGCTGATTTATTGGGCGAGCACCCGCGAAAGCGGCGTGCCGGATCGCTTTAGCGGCAGCGGCAGCGGCACCGATTTTACCCTGACCATTAGCAGCCTGCAGGCGGAAGATGTGGCGGTGTATTATTGCCATCAGTATATGAGCAGCTATACCTTTGGCCAGGGCACCAAACTGGAAATTAAA

TABLE 1B mAb2-3 IgG4 Variable Region amino acid sequencesV_(H) chain of mAb2-3 IgG4 (SEQ ID NO: 2)QVQLVQSGAEVKKPGASVKVSCKASGYTFASAWMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARST YYRPLDYWGQGTLVTVSSV_(L) chain of mAb2-3 IgG4 (SEQ ID NO: 4)DIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYMSS YTFGQGTKLEIK

TABLE 2A Antibody 1-44 Variable Region nucleic acid sequencesV_(H) chain of 1-44 (SEQ ID NO: 15)CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGAGCTTCCGTCAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCGCCAGCCAATGGATGCACTGGATGCGGCAGGCACCTGGACAGGGCCTCGAATGGATCGGCTGGATCAACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGGCAGGGCCACCCTGACCGTGGACACCAGCACCAACACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGAAGCACCTGGTACCGGCCGCTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAG CAGCV_(L) chain of 1-44 (SEQ ID NO: 17)GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGGGCCACCATCAACTGCAAGAGCAGCCAGAGCATCCTGTACAGCAGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCCGGGAGAGCGGCGTGCCCGACCGGTTTAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCACCAGTACATCAGCAGCTACACCTTCGGCCAGGGCACAAAGCTGGAAATCAAG

TABLE 2B Antibody 1-44 Variable Region amino acid sequencesV_(H) chain of 1-44 (SEQ ID NO: 16)QVQLVQSGAEVKKPGASVKVSCKASGYTFASQWMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARST WYRPLDYWGQGTLVTVSSV_(L) chain of 1-44 (SEQ ID NO: 18)DIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYISS YTFGQGTKLEIK

TABLE 3A Antibody 1-49 Variable Region nucleic acid sequencesV_(H) chain of 1-49 (SEQ ID NO: 19)CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGAGCTTCCGTCAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCGCCAGCAGCTGGATGCACTGGATGCGGCAGGCACCTGGACAGGGCCTCGAATGGATCGGCTGGATCAACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGGCAGGGCCACCCTGACCGTGGACACCAGCACCAACACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGAAGCACGTGGTATCGGCCGAATGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAG CAGCV_(L) chain of 1-49 (SEQ ID NO: 21)GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGGGCCACCATCAACTGCAAGAGCAGCCAGAGCATCCTGTACAGCAGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCCGGGAGAGCGGCGTGCCCGACCGGTTTAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCACCAGTACAAAAGCAGCTACACCTTCGGCCAGGGCACAAAGCTGGAAATCAAG

TABLE 3B Antibody 1-49 Variable Region amino acid sequencesV_(H) chain of 1-49 (SEQ ID NO: 20)QVQLVQSGAEVKKPGASVKVSCKASGYTFASSWMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARST WYRPNDYWGQGTLVTVSSV_(L) chain of 1-49 (SEQ ID NO: 22)DIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYKSS YTFGQGTKLEIK

TABLE 4A Antibody 2-1 Variable Region nucleic acid sequencesV_(H) chain of 2-1 (SEQ ID NO: 23)CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGAGCTTCCGTCAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCGCCAGCAGCTGGATGCACTGGATGCGGCAGGCACCTGGACAGGGCCTCGAATGGATCGGCTGGATCAACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGGCAGGGCCACCCTGACCGTGGACACCAGCACCAACACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGAACCACCCGTTATCGGCCCCTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAG CAGCV_(L) chain of 2-1 (SEQ ID NO: 25)GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGGGCCACCATCAACTGCAAGAGCAGCCAGAGCATCCTGTACAGCAGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCCGGGAGAGCGGCGTGCCCGACCGGTTTAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCACCAGTACCGTAGCAGCTACACCTTCGGCCAGGGCACAAAGCTGGAAATCAAG

TABLE 4B Antibody 2-1 Variable Region amino acid sequencesV_(H) chain of 2-1 (SEQ ID NO: 24)QVQLVQSGAEVKKPGASVKVSCKASGYTFASSWMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARTT RYRPLDYWGQGTLVTVSSV_(L) chain of 2-1 (SEQ ID NO: 26)DIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYRSS YTFGQGTKLEIK

TABLE 5A Antibody 2-2 Variable Region nucleic acid sequencesV_(H) chain of 2-2 (SEQ ID NO: 27)CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGAGCTTCCGTCAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCGCCAGCCAATATATGCACTGGATGCGGCAGGCACCTGGACAGGGCCTCGAATGGATCGGCTGGATCAACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGGCAGGGCCACCCTGACCGTGGACACCAGCACCAACACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGACTGACCTATTATCGGCCGCCGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAG CAGCV_(L) chain of 2-2 (SEQ ID NO: 29)GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGGGCCACCATCAACTGCAAGAGCAGCCAGAGCATCCTGTACAGCAGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCCGGGAGAGCGGCGTGCCCGACCGGTTTAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCACCAGTACTATAGCAGCTACACCTTCGGCCAGGGCACAAAGCTGGAAATCAAG

TABLE 5B Antibody 2-2 Variable Region amino acid sequencesV_(H) chain of 2-2 (SEQ ID NO: 28)QVQLVQSGAEVKKPGASVKVSCKASGYTFASQYMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARLT YYRPPDYWGQGTLVTVSSV_(L) chain of 2-2 (SEQ ID NO: 30)DIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYYSS YTFGQGTKLEIK

TABLE 6A huCCR Variable Region nucleic acid sequencesV_(H) chain of huCCR (SEQ ID NO: 43)CAGGTGCAGCTGGTGCAGAGCGGAGCCGAGGTGAAGAAGCCTGGAGCTTCCGTCAAGGTGTCCTGCAAGGCCAGCGGCTACACCTTCGCCAGCTACTACATGCACTGGATGCGGCAGGCACCTGGACAGGGCCTCGAATGGATCGGCTGGATCAACCCCGGCAACGTGAACACCAAGTACAACGAGAAGTTCAAGGGCAGGGCCACCCTGACCGTGGACACCAGCACCAACACCGCCTACATGGAACTGAGCAGCCTGCGGAGCGAGGACACCGCCGTGTACTACTGCGCCAGAAGCACCTACTACCGGCCCCTGGACTACTGGGGCCAGGGCACCCTGGTGACCGTGAG CAGCV_(L) chain of huCCR (SEQ ID NO: 45)GACATCGTGATGACCCAGAGCCCCGACAGCCTGGCCGTGAGCCTGGGCGAGCGGGCCACCATCAACTGCAAGAGCAGCCAGAGCATCCTGTACAGCAGCAACCAGAAGAACTACCTGGCCTGGTATCAGCAGAAGCCCGGCCAGAGCCCCAAGCTGCTGATCTACTGGGCCAGCACCCGGGAGAGCGGCGTGCCCGACCGGTTTAGCGGCAGCGGCTCCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGGCCGAGGACGTGGCCGTGTACTACTGCCACCAGTACCTGAGCAGCTACACCTTCGGCCAGGGCACAAAGCTGGAAATCAAG

TABLE 6B huCCR Variable Region, amino acid sequencesV_(H) chain of huCCR (SEQ ID NO: 44)QVQLVQSGAEVKKPGASVKVSCKASGYTFASYYMHWMRQAPGQGLEWIGWINPGNVNTKYNEKFKGRATLTVDTSTNTAYMELSSLRSEDTAVYYCARST YYRPLDYWGQGTLVTVSSV_(L) chain of huCCR (SEQ ID NO: 46)DIVMTQSPDSLAVSLGERATINCKSSQSILYSSNQKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCHQYLSS YTFGQGTKLEIK

TABLE 7A IgG4 Isotype Region nucleic acid sequencesIgG4 Isotype Region nucleic acids (SEQ ID NO: 5)GCGAGCACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGTGCAGCCGCAGCACCAGCGAAAGCACCGCGGCGCTGGGCTGCCTGGTGAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGCGCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCCTGGGCACCAAAACCTATACCTGCAACGTGGATCATAAACCGAGCAACACCAAAGTGGATAAACGCGTGGAAAGCAAATATGGCCCGCCGTGCCCGAGCTGCCCGGCGCCGGAATTTCTGGGCGGCCCGAGCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATTAGCCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCAGGAAGATCCGGAAGTGCAGTTTAACTGGTATGTGGATGGCGTGGAAGTGCATAACGCGAAAACCAAACCGCGCGAAGAACAGTTTAACAGCACCTATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAAGAATATAAATGCAAAGTGAGCAACAAAGGCCTGCCGAGCAGCATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACCGCAGGTGTATACCCTGCCGCCGAGCCCGGAAGAAATGACCAAAAACCAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATATTGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAACTATAAAACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTTTTCTGTATAGCCGCCTGACCGTGGATAAAAGCCGCTGGCAGGAAGGCAACGTGTTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAGCCTGAGCCTGAGCCTGGGCAAA

TABLE 7B IgG4 Isotype Region amino acid sequencesIgG4 Isotype Region amino acid sequences (SEQ ID NO: 6)ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSPEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

TABLE 8A IgG4 with stabilized IgG4 core hinge, nucleic acid sequencesIgG4 Isotype Region nucleic acids (SEQ ID NO: 7)ACCAAAGGCCCGAGCGTGTTTCCGCTGGCGCCGTGCAGCCGCAGCACCAGCGAAAGCACCGCGGCGCTGGGCTGCCTGGTGAAAGATTATTTTCCGGAACCGGTGACCGTGAGCTGGAACAGCGGCGCGCTGACCAGCGGCGTGCATACCTTTCCGGCGGTGCTGCAGAGCAGCGGCCTGTATAGCCTGAGCAGCGTGGTGACCGTGCCGAGCAGCAGCCTGGGCACCAAAACCTATACCTGCAACGTGGATCATAAACCGAGCAACACCAAAGTGGATAAACGCGTGGAAAGCAAATATGGCCCGCCGTGCCCGCCGTGCCCGGCGCCGGAATTTCTGGGCGGCCCGAGCGTGTTTCTGTTTCCGCCGAAACCGAAAGATACCCTGATGATTAGCCGCACCCCGGAAGTGACCTGCGTGGTGGTGGATGTGAGCCAGGAAGATCCGGAAGTGCAGTTTAACTGGTATGTGGATGGCGTGGAAGTGCATAACGCGAAAACCAAACCGCGCGAAGAACAGTTTAACAGCACCTATCGCGTGGTGAGCGTGCTGACCGTGCTGCATCAGGATTGGCTGAACGGCAAAGAATATAAATGCAAAGTGAGCAACAAAGGCCTGCCGAGCAGCATTGAAAAAACCATTAGCAAAGCGAAAGGCCAGCCGCGCGAACCGCAGGTGTATACCCTGCCGCCGAGCCCGGAAGAAATGACCAAAAACCAGGTGAGCCTGACCTGCCTGGTGAAAGGCTTTTATCCGAGCGATATTGCGGTGGAATGGGAAAGCAACGGCCAGCCGGAAAACAACTATAAAACCACCCCGCCGGTGCTGGATAGCGATGGCAGCTTTTTTCTGTATAGCAAACTGACCGTGGATAAAAGCCGCTGGCAGGAAGGCAACGTGTTTAGCTGCAGCGTGATGCATGAAGCGCTGCATAACCATTATACCCAGAAAAGCCTGAGCCTGAGCCTGGGCAAA

TABLE 8B IgG4 with stabilized IgG4 core hinge, amino acid sequencesIgG4 Isotype Region amino acid sequences (SEQ ID NO: 8)TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLEPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSPEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFELYSKLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

The amino acid sequences of the heavy and light chain complementaritydetermining regions of selected antibodies are shown in Table 9 below.

TABLE 9 Amino Acid Sequences of Heavy and Light Chains. VariableAntibody region CDR1 CDR2 CDR3 Mouse 1567 VH GYTFASYY INPGNVNT STYYRPLDY(SEQ ID NO: 31) (SEQ ID NO: 11) (SEQ ID NO: 13) Humanized 1567 VHGYTFASYY INPGNVNT STYYRPLDY (SEQ ID NO: 31) (SEQ ID NO: 11)(SEQ ID NO: 13) Ab1-44 VH GYTFASQW INPGNVNT STWYRPLDY (SEQ ID NO: 32)(SEQ ID NO: 11) (SEQ ID NO: 34) Ab1-49 VH GYTFASSW INPGNVNT STWYRPNDY(SEQ ID NO: 33) (SEQ ID NO: 11) (SEQ ID NO: 35) Ab2-1 VH GYTFASSWINPGNVNT TTRYRPLDY (SEQ ID NO: 33) (SEQ ID NO: 11) (SEQ ID NO: 36) Ab2-2VH GYTFASSW INPGNVNT LTYYRPPDY (SEQ ID NO: 33) (SEQ ID NO: 11)(SEQ ID NO: 37) Ab2-3 VH GYTFASAW INPGNVNT STYYRPLDY (SEQ ID NO: 9)(SEQ ID NO: 11) (SEQ ID NO: 13) Mouse 1567 VL QSILYSSNQKNY WASTREHQYLSSYT (SEQ ID NO: 10) (SEQ ID NO: 12) (SEQ ID NO: 38) Humanized 1567VL QSILYSSNQKNY WASTRE HQYLSSYT (SEQ ID NO: 10) (SEQ ID NO: 12)(SEQ ID NO: 38) Ab1-44 VL QSILYSSNQKNY WASTRE HQYISSYT (SEQ ID NO: 10)(SEQ ID NO: 12) (SEQ ID NO: 39) Ab1-49 VL QSILYSSNQKNY WASTRE HQYKSSYT(SEQ ID NO: 10) (SEQ ID NO: 12) (SEQ ID NO: 40) Ab2-1 VL QSILYSSNQKNYWASTRE HQYRSSYT (SEQ ID NO: 10) (SEQ ID NO: 12) (SEQ ID NO: 41) Ab2-2 VLQSILYSSNQKNY WASTRE HQYYSSYT (SEQ ID NO: 10) (SEQ ID NO: 12)(SEQ ID NO: 42) Ab2-3 VL QSILYSSNQKNY WASTRE HQYMSSYT (SEQ ID NO: 10)(SEQ ID NO: 12) (SEQ ID NO: 14)

As described supra, CCR4-IgG4 of the present invention modulates T cellactivity. In some aspects, administration of CCR4-IgG4 reversesregulatory T-cell-mediated suppression of effector T cell proliferation.Specifically, treatment with CCR4-IgG4 stimulates or increasesproliferation of effector T cells (Teff), without stimulating theproliferation of regulatory T cells (Treg). Effector T cells consist offour distinct populations, as classified by CD45RA and CCR7 expressionprofiles: T-different types (Tdiff), naïve T cells (Tnaive), centralmemory T cells (Tcm) and effector memory T cells (Tem). The CCR4-IgG4 ofthe present invention can stimulate or increase the proliferation of anyof the Teff populations. In some aspects, increasing proliferation ofeffector T cells increases antigen-specific T cell activity to augmentan immune response to an antigen. In some aspects, augmenting effectorT-cell-mediated immune response may contribute to inhibition oftumorigenesis or reduction in tumor size.

In other aspects, CCR4-IgG4 modulates T cell cytokine production andsecretion. For example, administration of CCR4-IgG4 specificallyincreases IFN-gamma (IFNγ) production and release from T cells. In otheraspects, administration of CCR4-IgG4 may not affect IL-10 or IL-4release. In another aspect, administration of CCR4-IgG4 may not affect,or may slightly reduce TGF-beta release. Cytokine release profiles mayindicate the specific T cell population activated by treatment withCCR4-IgG4, as IFNγ secretion is a characteristic of Th1 cells (T-helpertype 1 cells), while TGF-beta and IL-10 secretion is characteristic ofregulatory T cells and IL-4 is released by Th2 (T helper type 2 cells).In some aspects, CCR4-IgG4 stimulates T cell activity, wherein the Tcells are Th1 cells. In some embodiments, CCR4-IgG4 stimulates secretionof IFNγ and decreases or does not change secretion of TGF-β, IL-10 orIL-4.

Antibodies

As used herein, the term “antibody” refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. By “specifically binds” or“immunoreacts with” is meant that the antibody reacts with one or moreantigenic determinants of the desired antigen and does not react withother polypeptides. Antibodies include, but are not limited to,polyclonal, monoclonal, chimeric, dAb (domain antibody), single chain,F_(ab), F_(ab′) and F_((ab′)2) fragments, scFvs, and F_(ab) expressionlibraries.

A single chain Fv (“scFv”) polypeptide molecule is a covalently linkedV_(H):V_(L) heterodimer, which can be expressed from a gene fusionincluding V_(H)- and V_(L)-encoding genes linked by a peptide-encodinglinker. (See Huston et al. (1988) Proc Nat Acad Sci USA85(16):5879-5883). A number of methods have been described to discernchemical structures for converting the naturally aggregated, butchemically separated, light and heavy polypeptide chains from anantibody V region into an scFv molecule, which will fold into a threedimensional structure substantially similar to the structure of anantigen-binding site. See, e.g., U.S. Pat. Nos. 5,091,513; 5,132,405;and 4,946,778.

Very large naïve human scFv libraries have been and can be created tooffer a large source of rearranged antibody genes against a plethora oftarget molecules. Smaller libraries can be constructed from individualswith infectious diseases in order to isolate disease-specificantibodies. (See Barbas et al., Proc. Natl. Acad. Sci. USA 89:9339-43(1992); Zebedee et al., Proc. Natl. Acad. Sci. USA 89:3175-79 (1992)).

In general, antibody molecules obtained from humans relate to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain. The term“antigen-binding site,” or “binding portion” refers to the part of theimmunoglobulin molecule that participates in antigen binding. Theantigen binding site is formed by amino acid residues of the N-terminalvariable (“V”) regions of the heavy (“H”) and light (“L”) chains. Threehighly divergent stretches within the V regions of the heavy and lightchains, referred to as “hypervariable regions,” are interposed betweenmore conserved flanking stretches known as “framework regions,” or“FRs”. Thus, the term “FR” refers to amino acid sequences which arenaturally found between, and adjacent to, hypervariable regions inimmunoglobulins. In an antibody molecule, the three hypervariableregions of a light chain and the three hypervariable regions of a heavychain are disposed relative to each other in three dimensional space toform an antigen-binding surface. The antigen-binding surface iscomplementary to the three-dimensional surface of a bound antigen, andthe three hypervariable regions of each of the heavy and light chainsare referred to as “complementarity-determining regions,” or “CDRs.”

As used herein, the term “epitope” includes any protein determinantcapable of specific binding to an immunoglobulin, a scFv, or a T-cellreceptor. Epitopic determinants usually consist of chemically activesurface groupings of molecules such as amino acids or sugar side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics. For example, antibodies maybe raised against N-terminal or C-terminal peptides of a polypeptide.

As used herein, the terms “immunological binding,” and “immunologicalbinding properties” refer to the non-covalent interactions of the typewhich occur between an immunoglobulin molecule and an antigen for whichthe immunoglobulin is specific. The strength, or affinity ofimmunological binding interactions can be expressed in terms of thedissociation constant (K_(d)) of the interaction, wherein a smallerK_(d) represents a greater affinity Immunological binding properties ofselected polypeptides can be quantified using methods well known in theart. One such method entails measuring the rates of antigen-bindingsite/antigen complex formation and dissociation, wherein those ratesdepend on the concentrations of the complex partners, the affinity ofthe interaction, and geometric parameters that equally influence therate in both directions. Thus, both the “on rate constant” (K_(on)) andthe “off rate constant” (K_(off)) can be determined by calculation ofthe concentrations and the actual rates of association and dissociation.(See Nature 361:186-87 (1993)). The ratio of K_(off)/K_(on) enables thecancellation of all parameters not related to affinity, and is equal tothe dissociation constant K_(d). (See, generally, Davies et al. (1990)Annual Rev Biochem 59:439-473). An antibody of the present invention issaid to specifically bind to a CCR4 epitope when the equilibrium bindingconstant (K_(d)) is ≤1 μM, preferably ≤100 nM, more preferably ≤10 nM,and most preferably ≤100 pM to about 1 pM, as measured by assays such asradioligand binding assays or similar assays known to those skilled inthe art.

A CCR4 protein of the invention, or a derivative, fragment, analog,homolog or ortholog thereof, may be utilized as an immunogen in thegeneration of antibodies that immunospecifically bind these proteincomponents.

Those skilled in the art will recognize that it is possible todetermine, without undue experimentation, if a human monoclonal antibodyhas the same specificity as a human monoclonal antibody of the inventionby ascertaining whether the former prevents the latter from binding toCCR4. If the human monoclonal antibody being tested competes with thehuman monoclonal antibody of the invention, as shown by a decrease inbinding by the human monoclonal antibody of the invention, then it islikely that the two monoclonal antibodies bind to the same, or to aclosely related, epitope.

Another way to determine whether a human monoclonal antibody has thespecificity of a human monoclonal antibody of the invention is topre-incubate the human monoclonal antibody of the invention with theCCR4 protein, with which it is normally reactive, and then add the humanmonoclonal antibody being tested to determine if the human monoclonalantibody being tested is inhibited in its ability to bind CCR4. If thehuman monoclonal antibody being tested is inhibited then, in alllikelihood, it has the same, or functionally equivalent, epitopespecificity as the monoclonal antibody of the invention. Screening ofhuman monoclonal antibodies of the invention, can be also carried out byutilizing CCR4 and determining whether the test monoclonal antibody isable to neutralize CCR4.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs ororthologs thereof (See, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference).

Antibodies can be purified by well-known techniques, such as affinitychromatography using protein A or protein G, which provide primarily theIgG fraction of immune serum. Subsequently, or alternatively, thespecific antigen which is the target of the immunoglobulin sought, or anepitope thereof, may be immobilized on a column to purify the immunespecific antibody by immunoaffinity chromatography. Purification ofimmunoglobulins is discussed, for example, by D. Wilkinson (TheScientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14,No. 8 (Apr. 17, 2000), pp. 25-28).

The term “monoclonal antibody” or “mAb” or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell (Goding, MonoclonalAntibodies: Principles and Practice, Academic Press, (1986) pp. 59-103)Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently,support stable high level expression of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. More preferred immortalized cell lines are murine myeloma lines,which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies. (See Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques andApplications, Marcel Dekker, Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then beassayed for the presence of monoclonal antibodies directed against theantigen. Preferably, the binding specificity of monoclonal antibodiesproduced by the hybridoma cells is determined by immunoprecipitation orby an in vitro binding assay, such as radioimmunoassay (RIA) orenzyme-linked immunoabsorbent assay (ELISA). Such techniques and assaysare known in the art. The binding affinity of the monoclonal antibodycan, for example, be determined by the Scatchard analysis of Munson andPollard, Anal. Biochem., 107:220 (1980). Moreover, in therapeuticapplications of monoclonal antibodies, it is important to identifyantibodies having a high degree of specificity and a high bindingaffinity for the target antigen.

After the desired hybridoma cells are identified, the clones can besubcloned by limiting dilution procedures and grown by standard methods.(See Goding, Monoclonal Antibodies: Principles and Practice, AcademicPress, (1986) pp. 59-103). Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-1640medium. Alternatively, the hybridoma cells can be grown in vivo asascites in a mammal.

The monoclonal antibodies secreted by the subclones can be isolated orpurified from the culture medium or ascites fluid by conventionalimmunoglobulin purification procedures such as, for example, proteinA-Sepharose, hydroxylapatite chromatography, gel electrophoresis,dialysis, or affinity chromatography.

Monoclonal antibodies can also be made by recombinant DNA methods, suchas those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells. The DNA also can be modified, for example, bysubstituting the coding sequence for human heavy and light chainconstant domains in place of the homologous murine sequences (see U.S.Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or bycovalently joining to the immunoglobulin coding sequence all or part ofthe coding sequence for a non-immunoglobulin polypeptide. Such anon-immunoglobulin polypeptide can be substituted for the constantdomains of an antibody of the invention, or can be substituted for thevariable domains of one antigen-combining site of an antibody of theinvention to create a chimeric bivalent antibody.

Fully human antibodies are antibody molecules in which the entiresequence of both the light chain and the heavy chain, including theCDRs, arise from human genes. Such antibodies are termed “humanizedantibodies”, “human antibodies”, or “fully human antibodies” herein.Human monoclonal antibodies can be prepared by using trioma technique;the human B-cell hybridoma technique (see Kozbor, et al., 1983 ImmunolToday 4: 72); and the EBV hybridoma technique to produce humanmonoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIESAND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96). Human monoclonalantibodies may be utilized and may be produced by using human hybridomas(see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or bytransforming human B-cells with Epstein Barr Virus in vitro (see Cole,et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss,Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries. (See Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)). Similarly, human antibodies can be made by introducinghuman immunoglobulin loci into transgenic animals, e.g., mice in whichthe endogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368, 812-13 (1994); Fishwild et al, NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); and Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096. This animal produces Bcells which secrete fully human immunoglobulins. The antibodies can beobtained directly from the animal after immunization with an immunogenof interest, as, for example, a preparation of a polyclonal antibody, oralternatively from immortalized B cells derived from the animal, such ashybridomas producing monoclonal antibodies. Additionally, the genesencoding the immunoglobulins with human variable regions can berecovered and expressed to obtain the antibodies directly, or can befurther modified to obtain analogs of antibodies such as, for example,single chain Fv (scFv) molecules.

An example of a method of producing a nonhuman host, exemplified as amouse, lacking expression of an endogenous immunoglobulin heavy chain isdisclosed in U.S. Pat. No. 5,939,598. It can be obtained by a method,which includes deleting the J segment genes from at least one endogenousheavy chain locus in an embryonic stem cell to prevent rearrangement ofthe locus and to prevent formation of a transcript of a rearrangedimmunoglobulin heavy chain locus, the deletion being effected by atargeting vector containing a gene encoding a selectable marker; andproducing from the embryonic stem cell a transgenic mouse whose somaticand germ cells contain the gene encoding the selectable marker.

One method for producing an antibody of interest, such as a humanantibody, is disclosed in U.S. Pat. No. 5,916,771. This method includesintroducing an expression vector that contains a nucleotide sequenceencoding a heavy chain into one mammalian host cell in culture,introducing an expression vector containing a nucleotide sequenceencoding a light chain into another mammalian host cell, and fusing thetwo cells to form a hybrid cell. The hybrid cell expresses an antibodycontaining the heavy chain and the light chain.

In a further improvement on this procedure, a method for identifying aclinically relevant epitope on an immunogen and a correlative method forselecting an antibody that binds immunospecifically to the relevantepitope with high affinity, are disclosed in PCT publication WO99/53049.

The antibody can be expressed by a vector containing a DNA segmentencoding the single chain antibody described above.

These can include vectors, liposomes, naked DNA, adjuvant-assisted DNA,gene gun, catheters, etc. Vectors include chemical conjugates such asdescribed in WO 93/64701, which has targeting moiety (e.g. a ligand to acellular surface receptor), and a nucleic acid binding moiety (e.g.polylysine), viral vector (e.g. a DNA or RNA viral vector), fusionproteins such as described in PCT/US 95/02140 (WO 95/22618) which is afusion protein containing a target moiety (e.g. an antibody specific fora target cell) and a nucleic acid binding moiety (e.g. a protamine),plasmids, phage, etc. The vectors can be chromosomal, non-chromosomal orsynthetic.

Preferred vectors include viral vectors, fusion proteins and chemicalconjugates. Retroviral vectors include moloney murine leukemia viruses.DNA viral vectors are preferred. These vectors include pox vectors suchas orthopox or avipox vectors, herpesvirus vectors such as a herpessimplex I virus (HSV) vector (see Geller, A. I. et al., J. Neurochem,64:487 (1995); Lim, F., et al., in DNA Cloning: Mammalian Systems, D.Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller, A. I.et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., etal., Proc Natl. Acad. Sci USA 87:1149 (1990), Adenovirus Vectors (seeLeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat.Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) andAdeno-associated Virus Vectors (see Kaplitt, M. G. et al., Nat. Genet.8:148 (1994).

Pox viral vectors introduce the gene into the cells cytoplasm. Avipoxvirus vectors result in only a short term expression of the nucleicacid. Adenovirus vectors, adeno-associated virus vectors and herpessimplex virus (HSV) vectors are preferred for introducing the nucleicacid into neural cells. The adenovirus vector results in a shorter termexpression (about 2 months) than adeno-associated virus (about 4months), which in turn is shorter than HSV vectors. The particularvector chosen will depend upon the target cell and the condition beingtreated. The introduction can be by standard techniques, e.g. infection,transfection, transduction or transformation. Examples of modes of genetransfer include e.g., naked DNA, CaPO₄ precipitation, DEAE dextran,electroporation, protoplast fusion, lipofection, cell microinjection,and viral vectors.

The vector can be employed to target essentially any desired targetcell. For example, stereotaxic injection can be used to direct thevectors (e.g. adenovirus, HSV) to a desired location. Additionally, theparticles can be delivered by intracerebroventricular (icy) infusionusing a minipump infusion system, such as a SynchroMed Infusion System.A method based on bulk flow, termed convection, has also proveneffective at delivering large molecules to extended areas of the brainand may be useful in delivering the vector to the target cell. (See Boboet al., Proc. Natl. Acad. Sci. USA 91:2076-2080 (1994); Morrison et al.,Am. J. Physiol. 266:292-305 (1994)). Other methods that can be usedinclude catheters, intravenous, parenteral, intraperitoneal andsubcutaneous injection, and oral or other known routes ofadministration.

These vectors can be used to express large quantities of antibodies thatcan be used in a variety of ways. For example, to detect the presence ofCCR4 in a sample. The antibody can also be used to try to bind to anddisrupt a CCR4 activity.

Techniques can be adapted for the production of single-chain antibodiesspecific to an antigenic protein of the invention (see e.g., U.S. Pat.No. 4,946,778). In addition, methods can be adapted for the constructionof F_(ab) expression libraries (see e.g., Huse, et al., 1989 Science246: 1275-1281) to allow rapid and effective identification ofmonoclonal F_(ab) fragments with the desired specificity for a proteinor derivatives, fragments, analogs or homologs thereof. Antibodyfragments that contain the idiotypes to a protein antigen may beproduced by techniques known in the art including, but not limited to:(i) an F_((ab′)2) fragment produced by pepsin digestion of an antibodymolecule; (ii) an F_(ab) fragment generated by reducing the disulfidebridges of an F_((ab′)2) fragment; (iii) an F_(ab) fragment generated bythe treatment of the antibody molecule with papain and a reducing agentand (iv) F_(v) fragments.

Heteroconjugate antibodies are also within the scope of the presentinvention. Heteroconjugate antibodies are composed of two covalentlyjoined antibodies. Such antibodies have, for example, been proposed totarget immune system cells to unwanted cells (see U.S. Pat. No.4,676,980), and for treatment of HIV infection (see WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies can beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinscan be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980. Heteroconjugate antibodies may alsorefer to bi-specific antibodies, wherein a bi-specific antibody iscomposed of, for example, two covalently joined single chain antibodies,or scFvs, or two covalently joined variable heavy chain-variable lightchain dimers from two antibodies that recognize different antigens.

It can be desirable to modify the antibody of the invention with respectto effector function, so as to enhance, e.g., the effectiveness of theantibody in treating cancer. For example, cysteine residue(s) can beintroduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedcan have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). (See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992)). Alternatively,an antibody can be engineered that has dual Fc regions and can therebyhave enhanced complement lysis and ADCC capabilities. (See Stevenson etal., Anti-Cancer Drug Design, 3: 219-230 (1989)).

The invention also pertains to immunoconjugates comprising an antibodyconjugated to a cytotoxic agent such as a toxin (e.g., an enzymaticallyactive toxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate).

Enzymatically active toxins and fragments thereof that can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin, and the tricothecenes. A variety of radionuclides areavailable for the production of radioconjugated antibodies. Examplesinclude ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a varietyof bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. (See WO94/11026).

Those of ordinary skill in the art will recognize that a large varietyof possible moieties can be coupled to the resultant antibodies or toother molecules of the invention. (See, for example, “ConjugateVaccines”, Contributions to Microbiology and Immunology, J. M. Cruse andR. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entirecontents of which are incorporated herein by reference).

Coupling may be accomplished by any chemical reaction that will bind thetwo molecules so long as the antibody and the other moiety retain theirrespective activities. This linkage can include many chemicalmechanisms, for instance covalent binding, affinity binding,intercalation, coordinate binding and complexation. The preferredbinding is, however, covalent binding. Covalent binding can be achievedeither by direct condensation of existing side chains or by theincorporation of external bridging molecules. Many bivalent orpolyvalent linking agents are useful in coupling protein molecules, suchas the antibodies of the present invention, to other molecules. Forexample, representative coupling agents can include organic compoundssuch as thioesters, carbodiimides, succinimide esters, diisocyanates,glutaraldehyde, diazobenzenes and hexamethylene diamines. This listingis not intended to be exhaustive of the various classes of couplingagents known in the art but, rather, is exemplary of the more commoncoupling agents. (See Killen and Lindstrom, Jour. Immun 133:1335-2549(1984); Jansen et al., Immunological Reviews 62:185-216 (1982); andVitetta et al., Science 238:1098 (1987)). Preferred linkers aredescribed in the literature. (See, for example, Ramakrishnan, S. et al.,Cancer Res. 44:201-208 (1984) describing use of MBS(M-maleimidobenzoyl-N-hydroxysuccinimide ester). See also, U.S. Pat. No.5,030,719, describing use of halogenated acetyl hydrazide derivativecoupled to an antibody by way of an oligopeptide linker. Particularlypreferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride; (ii) SMPT(4-succinimidyloxycarbonyl-alpha-methyl-alpha-(2-pridyl-dithio)-toluene(Pierce Chem. Co., Cat. (21558G); (iii) SPDP (succinimidyl-6[3-(2-pyridyldithio) propionamido]hexanoate (Pierce Chem. Co., Cat#21651G); (iv) Sulfo-LC-SPDP (sulfosuccinimidyl 6[3-(2-pyridyldithio)-propianamide] hexanoate (Pierce Chem. Co. Cat.#2165-G); and (v) sulfo-NHS (N-hydroxysulfo-succinimide: Pierce Chem.Co., Cat. #24510) conjugated to EDC.

The linkers described above contain components that have differentattributes, thus leading to conjugates with differing physio-chemicalproperties. For example, sulfo-NHS esters of alkyl carboxylates are morestable than sulfo-NHS esters of aromatic carboxylates. NHS-estercontaining linkers are less soluble than sulfo-NHS esters. Further, thelinker SMPT contains a sterically hindered disulfide bond, and can formconjugates with increased stability. Disulfide linkages, are in general,less stable than other linkages because the disulfide linkage is cleavedin vitro, resulting in less conjugate available. Sulfo-NHS, inparticular, can enhance the stability of carbodimide couplings.Carbodimide couplings (such as EDC) when used in conjunction withsulfo-NHS, forms esters that are more resistant to hydrolysis than thecarbodimide coupling reaction alone.

In some embodiments, mutations are introduced to the constant regions ofthe mAb such that the antibody dependent cell-mediated cytotoxicity(ADCC) activity of the mAb is altered. For example, the mutation is anLALA mutation in the CH2 domain, wherein the leucines at positions 234and 235 of the Fc region is mutated to alanine, and abrogates binding byspecific Fc receptors. In one aspect, the mAb contains mutations on onescFv molecule of the heterodimeric mAb, which reduces the ADCC activity.In another aspect, the mAb contains mutations on both chains of theheterodimeric mAb, which completely ablates the ADCC activity. Forexample, the mutations introduced one or both scFv molecules of the mAbare LALA mutations in the CH2 domain. These mAbs with variable ADCCactivity can be optimized such that the mAbs exhibits maximal selectivekilling towards cells that express one antigen that is recognized by themAb, however exhibits minimal killing towards the second antigen that isrecognized by the mAb.

The antibodies disclosed herein can also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

Particularly useful liposomes can be generated by the reverse-phaseevaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction.

Use of Antibodies Against CCR4

Methods for the screening of antibodies that possess the desiredspecificity include, but are not limited to, enzyme linked immunosorbentassay (ELISA) and other immunologically mediated techniques known withinthe art.

Antibodies directed against a CCR4 protein (or a fragment thereof) maybe used in methods known within the art relating to the localizationand/or quantitation of a CCR4 protein (e.g., for use in measuring levelsof the CCR4 protein within appropriate physiological samples, for use indiagnostic methods, for use in imaging the protein, and the like). In agiven embodiment, antibodies specific to a CCR4 protein, or derivative,fragment, analog or homolog thereof, that contain the antibody derivedantigen binding domain, are utilized as pharmacologically activecompounds (referred to hereinafter as “Therapeutics”).

An antibody specific for a CCR4 protein of the invention can be used toisolate a CCR4 polypeptide by standard techniques, such asimmunoaffinity, chromatography or immunoprecipitation. Antibodiesdirected against a CCR4 protein (or a fragment thereof) can be useddiagnostically to monitor protein levels in tissue as part of a clinicaltesting procedure, e.g., to, for example, determine the efficacy of agiven treatment regimen. Detection can be facilitated by coupling (i.e.,physically linking) the antibody to a detectable substance. Examples ofdetectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,and radioactive materials. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, β-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin, and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ³⁵S or ³H.

Antibodies of the invention, including polyclonal, monoclonal, humanizedand fully human antibodies, may used as therapeutic agents. Such agentswill generally be employed to treat or prevent cancer in a subject,increase vaccine efficiency or augment a natural immune response. Anantibody preparation, preferably one having high specificity and highaffinity for its target antigen, is administered to the subject and willgenerally have an effect due to its binding with the target.Administration of the antibody may abrogate or inhibit or interfere withan activity of the CCR4 protein.

A therapeutically effective amount of an antibody of the inventionrelates generally to the amount needed to achieve a therapeuticobjective. As noted above, this may be a binding interaction between theantibody and its target antigen that, in certain cases, interferes withthe functioning of the target. The amount required to be administeredwill furthermore depend on the binding affinity of the antibody for itsspecific antigen, and will also depend on the rate at which anadministered antibody is depleted from the free volume other subject towhich it is administered. Common ranges for therapeutically effectivedosing of an antibody or antibody fragment of the invention may be, byway of nonlimiting example, from about 0.1 mg/kg body weight to about 50mg/kg body weight. Common dosing frequencies may range, for example,from twice daily to once a week.

Antibodies specifically binding a CCR4 protein or a fragment thereof ofthe invention, as well as other molecules identified by the screeningassays disclosed herein, can be administered for the treatment of canceror other proliferative disorders in the form of pharmaceuticalcompositions. Principles and considerations involved in preparing suchcompositions, as well as guidance in the choice of components areprovided, for example, in Remington: The Science And Practice OfPharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub. Co.,Easton, Pa., 1995; Drug Absorption Enhancement: Concepts, Possibilities,Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa.,1994; and Peptide And Protein Drug Delivery (Advances In ParenteralSciences, Vol. 4), 1991, M. Dekker, New York.

Where antibody fragments are used, the smallest inhibitory fragment thatspecifically binds to the binding domain of the target protein ispreferred. For example, based upon the variable-region sequences of anantibody, peptide molecules can be designed that retain the ability tobind the target protein sequence. Such peptides can be synthesizedchemically and/or produced by recombinant DNA technology. (See, e.g.,Marasco et al., Proc. Natl. Acad. Sci. USA, 90: 7889-7893 (1993)). Theformulation can also contain more than one active compound as necessaryfor the particular indication being treated, preferably those withcomplementary activities that do not adversely affect each other.Alternatively, or in addition, the composition can comprise an agentthat enhances its function, such as, for example, a cytotoxic agent,cytokine, chemotherapeutic agent, or growth-inhibitory agent. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients can also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacrylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Sustained-release preparations can be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

An antibody according to the invention can be used as an agent fordetecting the presence of CCR4 (or a protein or a protein fragmentthereof) in a sample. Preferably, the antibody contains a detectablelabel. Antibodies can be polyclonal, or more preferably, monoclonal. Anintact antibody, or a fragment thereof (e.g., F_(ab), scFv, orF_((ab)2)) can be used. The term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject, as well as tissues, cells and fluids present within a subject.Included within the usage of the term “biological sample”, therefore, isblood and a fraction or component of blood including blood serum, bloodplasma, or lymph. That is, the detection method of the invention can beused to detect an analyte mRNA, protein, or genomic DNA in a biologicalsample in vitro as well as in vivo. For example, in vitro techniques fordetection of an analyte mRNA includes Northern hybridizations and insitu hybridizations. In vitro techniques for detection of an analyteprotein include enzyme linked immunosorbent assays (ELISAs), Westernblots, immunoprecipitations, and immunofluorescence. In vitro techniquesfor detection of an analyte genomic DNA include Southern hybridizations.Procedures for conducting immunoassays are described, for example in“ELISA: Theory and Practice: Methods in Molecular Biology”, Vol. 42, J.R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; “Immunoassay”, E.Diamandis and T. Christopoulus, Academic Press, Inc., San Diego, Calif.,1996; and “Practice and Theory of Enzyme Immunoassays”, P. Tijssen,Elsevier Science Publishers, Amsterdam, 1985. Furthermore, in vivotechniques for detection of an analyte protein include introducing intoa subject a labeled anti-analyte protein antibody. For example, theantibody can be labeled with a radioactive marker whose presence andlocation in a subject can be detected by standard imaging techniques.

Pharmaceutical Compositions

The antibodies or agents of the invention (also referred to herein as“active compounds”), and derivatives, fragments, analogs and homologsthereof, can be incorporated into pharmaceutical compositions suitablefor administration. Such compositions typically comprise the antibody oragent and a pharmaceutically acceptable carrier. As used herein, theterm “pharmaceutically acceptable carrier” is intended to include anyand all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in the most recent edition of Remington's PharmaceuticalSciences, a standard reference text in the field, which is incorporatedherein by reference. Preferred examples of such carriers or diluentsinclude, but are not limited to, water, saline, ringer's solutions,dextrose solution, and 5% human serum albumin. Liposomes and non-aqueousvehicles such as fixed oils may also be used. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfate; chelating agents such as ethylenediaminetetraacetic acid(EDTA); buffers such as acetates, citrates or phosphates, and agents forthe adjustment of tonicity such as sodium chloride or dextrose. The pHcan be adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Screening Methods

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., peptides, peptidomimetics, small molecules or otherdrugs) that modulate or otherwise interfere with a CCR4 activity. Alsoprovided are methods of identifying compounds useful to treat cancer.The invention also encompasses compounds identified using the screeningassays described herein.

For example, the invention provides assays for screening candidate ortest compounds which modulate the CCR4 carbonic anhydrase activity. Thetest compounds of the invention can be obtained using any of thenumerous approaches in combinatorial library methods known in the art,including: biological libraries; spatially addressable parallel solidphase or solution phase libraries; synthetic library methods requiringdeconvolution; the “one-bead one-compound” library method; and syntheticlibrary methods using affinity chromatography selection. The biologicallibrary approach is limited to peptide libraries, while the other fourapproaches are applicable to peptide, non-peptide oligomer or smallmolecule libraries of compounds. (See, e.g., Lam, 1997. Anticancer DrugDesign 12: 145).

A “small molecule” as used herein, is meant to refer to a compositionthat has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be, e.g., nucleic acids,peptides, polypeptides, peptidomimetics, carbohydrates, lipids or otherorganic or inorganic molecules. Libraries of chemical and/or biologicalmixtures, such as fungal, bacterial, or algal extracts, are known in theart and can be screened with any of the assays of the invention.

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt, et al., 1993. Proc. Natl.Acad. Sci. U.S.A. 90: 6909; Erb, et al., 1994. Proc. Natl. Acad. Sci.U.S.A. 91: 11422; Zuckermann, et al., 1994. J. Med. Chem. 37: 2678; Cho,et al., 1993. Science 261: 1303; Carrell, et al., 1994. Angew. Chem.Int. Ed. Engl. 33: 2059; Carell, et al., 1994. Angew. Chem. Int. Ed.Engl. 33: 2061; and Gallop, et al., 1994. J. Med. Chem. 37: 1233.

Libraries of compounds may be presented in solution (see e.g., Houghten,1992. Biotechniques 13: 412-421), or on beads (see Lam, 1991. Nature354: 82-84), on chips (see Fodor, 1993. Nature 364: 555-556), bacteria(see U.S. Pat. No. 5,223,409), spores (see U.S. Pat. No. 5,233,409),plasmids (see Cull, et al., 1992. Proc. Natl. Acad. Sci. USA 89:1865-1869) or on phage (see Scott and Smith, 1990. Science 249: 386-390;Devlin, 1990. Science 249: 404-406; Cwirla, et al., 1990. Proc. Natl.Acad. Sci. U.S.A. 87: 6378-6382; Felici, 1991. J. Mol. Biol. 222:301-310; and U.S. Pat. No. 5,233,409.).

In one embodiment, a candidate compound is introduced to anantibody-antigen complex and determining whether the candidate compounddisrupts the antibody-antigen complex, wherein a disruption of thiscomplex indicates that the candidate compound modulates an CCR4activity.

In another embodiment, at least one CCR4 protein is provided, which isexposed to at least one neutralizing monoclonal antibody. Formation ofan antibody-antigen complex is detected, and one or more candidatecompounds are introduced to the complex. If the antibody-antigen complexis disrupted following introduction of the one or more candidatecompounds, the candidate compounds is useful to treat cancer or aproliferative disease or disorder, particularly a renal proliferativedisorder.

Determining the ability of the test compound to interfere with ordisrupt the antibody-antigen complex can be accomplished, for example,by coupling the test compound with a radioisotope or enzymatic labelsuch that binding of the test compound to the antigen orbiologically-active portion thereof can be determined by detecting thelabeled compound in a complex. For example, test compounds can belabeled with ¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, andthe radioisotope detected by direct counting of radioemission or byscintillation counting. Alternatively, test compounds can beenzymatically-labeled with, for example, horseradish peroxidase,alkaline phosphatase, or luciferase, and the enzymatic label detected bydetermination of conversion of an appropriate substrate to product.

In one embodiment, the assay comprises contacting an antibody-antigencomplex with a test compound, and determining the ability of the testcompound to interact with the antigen or otherwise disrupt the existingantibody-antigen complex. In this embodiment, determining the ability ofthe test compound to interact with the antigen and/or disrupt theantibody-antigen complex comprises determining the ability of the testcompound to preferentially bind to the antigen or a biologically-activeportion thereof, as compared to the antibody.

In another embodiment, the assay comprises contacting anantibody-antigen complex with a test compound and determining theability of the test compound to modulate the antibody-antigen complex.Determining the ability of the test compound to modulate theantibody-antigen complex can be accomplished, for example, bydetermining the ability of the antigen to bind to or interact with theantibody, in the presence of the test compound.

Those skilled in the art will recognize that, in any of the screeningmethods disclosed herein, the antibody may be a CCR4 neutralizingantibody. Additionally, the antigen may be a CCR4 protein or a portionthereof (e.g., the CA domain).

The screening methods disclosed herein may be performed as a cell-basedassay or as a cell-free assay. In the case of cell-free assayscomprising the membrane-bound forms of the CCR4 proteins, it may bedesirable to utilize a solubilizing agent such that the membrane-boundform of the proteins is maintained in solution. Examples of suchsolubilizing agents include non-ionic detergents such asn-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate,3-(3-cholamidopropyl) dimethylamminiol-1-propane sulfonate (CHAPS), or3-(3-cholamidopropyl)dimethylamminiol-2-hydroxy-1-propane sulfonate(CHAPSO).

In more than one embodiment, it may be desirable to immobilize eitherthe antibody or the antigen to facilitate separation of complexed fromuncomplexed forms of one or both following introduction of the candidatecompound, as well as to accommodate automation of the assay. Observationof the antibody-antigen complex in the presence and absence of acandidate compound can be accomplished in any vessel suitable forcontaining the reactants. Examples of such vessels include microtiterplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided that adds a domain that allows one orboth of the proteins to be bound to a matrix. For example, GST-antibodyfusion proteins or GST-antigen fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, that are then combined withthe test compound, and the mixture is incubated under conditionsconducive to complex formation (e.g., at physiological conditions forsalt and pH). Following incubation, the beads or microtiter plate wellsare washed to remove any unbound components, the matrix immobilized inthe case of beads, complex determined either directly or indirectly.Alternatively, the complexes can be dissociated from the matrix, and thelevel of antibody-antigen complex formation can be determined usingstandard techniques.

Other techniques for immobilizing proteins on matrices can also be usedin the screening assays of the invention. For example, either theantibody or the antigen (e.g. the CCR4 protein or the CA domain thereof)can be immobilized utilizing conjugation of biotin and streptavidin.Biotinylated antibody or antigen molecules can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques well-known withinthe art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, other antibodies reactive with the antibody orantigen of interest, but which do not interfere with the formation ofthe antibody-antigen complex of interest, can be derivatized to thewells of the plate, and unbound antibody or antigen trapped in the wellsby antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using such other antibodiesreactive with the antibody or antigen.

The invention further pertains to novel agents identified by any of theaforementioned screening assays and uses thereof for treatments asdescribed herein.

Diagnostic Assays

Antibodies of the present invention can be detected by appropriateassays, e.g., conventional types of immunoassays. For example, asandwich assay can be performed in which a CCR4 protein or fragmentthereof (e.g., the CA domain) is affixed to a solid phase. Incubation ismaintained for a sufficient period of time to allow the antibody in thesample to bind to the immobilized polypeptide on the solid phase. Afterthis first incubation, the solid phase is separated from the sample. Thesolid phase is washed to remove unbound materials and interferingsubstances such as non-specific proteins which may also be present inthe sample. The solid phase containing the antibody of interest bound tothe immobilized polypeptide is subsequently incubated with a second,labeled antibody or antibody bound to a coupling agent such as biotin oravidin. This second antibody may be another anti-CCR4 antibody oranother antibody. Labels for antibodies are well-known in the art andinclude radionuclides, enzymes (e.g. maleate dehydrogenase, horseradishperoxidase, glucose oxidase, catalase), fluors (fluoresceinisothiocyanate, rhodamine, phycocyanin, fluorescarmine), biotin, and thelike. The labeled antibodies are incubated with the solid and the labelbound to the solid phase is measured. These and other immunoassays canbe easily performed by those of ordinary skill in the art.

The anti-CCR4 antibodies and scFv antibodies of the invention, whenjoined to a detectable moiety, provides a way for detecting “canceroustissue” or tissue subject to aberrant cell proliferation and thereforeat risk for cancer. In addition to tissue that becomes cancerous due toan in situ neoplasm, for example, the antibody-detectable moietyconjugates also provides a method of detecting cancerous metastatictissue present in distal organs and/or tissues. Thus such tissue may bedetected by contacting tissue suspected of being cancerous with theantibody-detectable moiety under appropriate conditions to cause thedetectable moiety to be detected in cancerous tissue, thereby detectingthe presence of cancerous tissue.

The detectable moieties can be conjugated directly to the antibodies orfragments, or indirectly by using, for example, a fluorescent secondaryantibody. Direct conjugation can be accomplished by standard chemicalcoupling of, for example, a fluorophore to the antibody or antibodyfragment, or through genetic engineering. Chimeras, or fusion proteinscan be constructed which contain an antibody or antibody fragmentcoupled to a fluorescent or bioluminescent protein. For example,Casadei, et al., describe a method of making a vector construct capableof expressing a fusion protein of aequorin and an antibody gene inmammalian cells.

As used herein, the term “labeled”, with regard to the probe orantibody, is intended to encompass direct labeling of the probe orantibody by coupling (i.e., physically linking) a detectable substanceto the probe or antibody, as well as indirect labeling of the probe orantibody by reactivity with another reagent that is directly labeled.Examples of indirect labeling include detection of a primary antibodyusing a fluorescently-labeled secondary antibody and end-labeling of aDNA probe with biotin such that it can be detected withfluorescently-labeled streptavidin. The term “biological sample” isintended to include tissues, cells and biological fluids isolated from asubject (such as a biopsy), as well as tissues, cells and fluids presentwithin a subject. That is, the detection method of the invention can beused to detect cancer, a cancer cell, or a cancer-associated cell (suchas a stromal cell associated with a tumor or cancer cell) in abiological sample in vitro as well as in vivo. For example, in vitrotechniques for detection of CCR4 include enzyme linked immunosorbentassays (ELISAs), Western blots, immunoprecipitations, andimmunofluorescence. Furthermore, in vivo techniques for detection ofCCR4 include introducing into a subject a labeled anti-CCR4 antibody.For example, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. In embodiments, the invention provides a non-invasive methodof detecting a tumor or cancer cell in a subject. The subject isadministered an antibody or scFv antibody of the invention, where theantibody is linked to a detectable moiety (i.e., any moiety capable ofbeing detected by, e.g., fluorescent, chemical, chemiluminescent,radioactive, or other means known in the art), the antibody is allowedto localize to the tumor then is detected by observation of thedetectable moiety.

In the case of “targeted” conjugates, that is, conjugates which containa targeting moiety—a molecule or feature designed to localize theconjugate within a subject or animal at a particular site or sites,localization refers to a state when an equilibrium between bound,“localized”, and unbound, “free” entities within a subject has beenessentially achieved. The rate at which such equilibrium is achieveddepends upon the route of administration. For example, a conjugateadministered by intravenous injection to localize thrombi may achievelocalization, or accumulation at the thrombi, within minutes ofinjection. On the other hand, a conjugate administered orally tolocalize an infection in the intestine may take hours to achievelocalization. Alternatively, localization may simply refer to thelocation of the entity within the subject or animal at selected timeperiods after the entity is administered. By way of another example,localization is achieved when an moiety becomes distributed followingadministration.

In all of the above cases, a reasonable estimate of the time to achievelocalization may be made by one skilled in the art. Furthermore, thestate of localization as a function of time may be followed by imagingthe detectable moiety (e.g., a light-emitting conjugate) according tothe methods of the invention, such as with a photodetector device. The“photodetector device” used should have a high enough sensitivity toenable the imaging of faint light from within a mammal in a reasonableamount of time, and to use the signal from such a device to construct animage.

In cases where it is possible to use light-generating moieties which areextremely bright, and/or to detect light-generating fusion proteinslocalized near the surface of the subject or animal being imaged, a pairof “night-vision” goggles or a standard high-sensitivity video camera,such as a Silicon Intensified Tube (SIT) camera (e.g., from HammamatsuPhotonic Systems, Bridgewater, N.J.), may be used. More typically,however, a more sensitive method of light detection is required.

In extremely low light levels the photon flux per unit area becomes solow that the scene being imaged no longer appears continuous. Instead,it is represented by individual photons which are both temporally andspatially distinct form one another. Viewed on a monitor, such an imageappears as scintillating points of light, each representing a singledetected photon. By accumulating these detected photons in a digitalimage processor over time, an image can be acquired and constructed. Incontrast to conventional cameras where the signal at each image point isassigned an intensity value, in photon counting imaging the amplitude ofthe signal carries no significance. The objective is to simply detectthe presence of a signal (photon) and to count the occurrence of thesignal with respect to its position over time.

At least two types of photodetector devices, described below, can detectindividual photons and generate a signal which can be analyzed by animage processor. Reduced-Noise Photodetection devices achievesensitivity by reducing the background noise in the photon detector, asopposed to amplifying the photon signal. Noise is reduced primarily bycooling the detector array. The devices include charge coupled device(CCD) cameras referred to as “backthinned”, cooled CCD cameras. In themore sensitive instruments, the cooling is achieved using, for example,liquid nitrogen, which brings the temperature of the CCD array toapproximately −120° C. “Backthinned” refers to an ultra-thin backplatethat reduces the path length that a photon follows to be detected,thereby increasing the quantum efficiency. A particularly sensitivebackthinned cryogenic CCD camera is the “TECH 512”, a series 200 cameraavailable from Photometrics, Ltd. (Tucson, Ariz.).

“Photon amplification devices” amplify photons before they hit thedetection screen. This class includes CCD cameras with intensifiers,such as microchannel intensifiers. A microchannel intensifier typicallycontains a metal array of channels perpendicular to and co-extensivewith the detection screen of the camera. The microchannel array isplaced between the sample, subject, or animal to be imaged, and thecamera. Most of the photons entering the channels of the array contact aside of a channel before exiting. A voltage applied across the arrayresults in the release of many electrons from each photon collision. Theelectrons from such a collision exit their channel of origin in a“shotgun” pattern, and are detected by the camera.

Even greater sensitivity can be achieved by placing intensifyingmicrochannel arrays in series, so that electrons generated in the firststage in turn result in an amplified signal of electrons at the secondstage. Increases in sensitivity, however, are achieved at the expense ofspatial resolution, which decreases with each additional stage ofamplification. An exemplary microchannel intensifier-based single-photondetection device is the C2400 series, available from Hamamatsu.

Image processors process signals generated by photodetector deviceswhich count photons in order to construct an image which can be, forexample, displayed on a monitor or printed on a video printer. Suchimage processors are typically sold as part of systems which include thesensitive photon-counting cameras described above, and accordingly, areavailable from the same sources. The image processors are usuallyconnected to a personal computer, such as an IBM-compatible PC or anApple Macintosh (Apple Computer, Cupertino, Calif.), which may or maynot be included as part of a purchased imaging system. Once the imagesare in the form of digital files, they can be manipulated by a varietyof image processing programs (such as “ADOBE PHOTOSHOP”, Adobe Systems,Adobe Systems, Mt. View, Calif.) and printed.

In one embodiment, the biological sample contains protein molecules fromthe test subject. One preferred biological sample is a peripheral bloodleukocyte sample isolated by conventional means from a subject.

The invention also encompasses kits for detecting the presence of CCR4or a CCR4-expressing cell in a biological sample. For example, the kitcan comprise: a labeled compound or agent capable of detecting a canceror tumor cell (e.g., an anti-CCR4 scFv or monoclonal antibody) in abiological sample; means for determining the amount of CCR4 in thesample; and means for comparing the amount of CCR4 in the sample with astandard. The standard is, in some embodiments, a non-cancer cell orcell extract thereof. The compound or agent can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect cancer in a sample.

Bi-Specific Antibodies

A bi-specific antibody (bsAb) is an antibody comprising two variabledomains or scFv units such that the resulting antibody recognizes twodifferent antigens. The present invention provides for bi-specificantibodies that recognize CCR4 and a second antigen. Exemplary secondantigens include tumor associated antigens, cytokines and cell surfacereceptors. In some embodiments, the second antigen can be CAIX (carbonicanhydrase IX, or G250, PD-L1, IL21, IL21R, HVEM, CD160, TIM3 or GALS.

A bi-specific antibody of the present invention comprises a heavy chainand a light chain combination or scFv of the CCR4-IgG4 antibodydisclosed herein.

Bi-specific antibodies of the present invention can be constructed usingmethods known art. In some embodiments, the bi-specific antibody is asingle polypeptide wherein two different heavy-light chain heterodimersor two different scFv antibodies, or fragments thereof, that eachrecognize a different antigen are joined by a long linker polypeptide,of sufficient length to allow intramolecular association between the twoscFv molecules to form a bi-specific antibody, with two heavy chains andtwo light chains. In one embodiment, one of the scFv moleculesrecognizes CCR4, for example, any of the scFv antibodies describedherein. In other embodiments, the bi-specific antibody consists of morethan one polypeptide, for example, two separate scFv antibodies, orfragments thereof, linked by covalent or non-covalent bonds, wherein oneof the scFv antibodies recognizes CCR4.

In one embodiment, the bi-specific antibody is constructed using the“knob into hole” method (Ridgway et al., Protein Eng 7:617-621 (1996)).In this method, the Ig heavy chains of the two different variabledomains are reduced to selectively break the heavy chain pairing whileretaining the heavy-light chain pairing. The two heavy-light chainheterodimers that recognize two different antigens are mixed to promoteheteroligation pairing, which is mediated through the engineered “knobinto holes” of the CH3 domains.

In another embodiment, the bi-specific antibody can be constructedthrough exchange of heavy-light chain heterodimers from two or moredifferent antibodies to generate a hybrid antibody where the firstheavy-light chain heterodimer recognizes CCR4 and the second heavy-lightchain heterodimer recognizes a second antigen. The mechanism forgenerating a bi-specific antibody consisting of two heavy-light chainheterodimers from two different antibodies is similar to the formationof human IgG4, which also functions as a bispecific molecule.Dimerization of IgG heavy chains is driven by intramolecular force, suchas the pairing the CH3 domain of each heavy chain and disulfide bridges.Presence of a specific amino acid in the CH3 domain (R409) has beenshown to promote dimer exchange and construction of the IgG4 molecules.Heavy chain pairing is also stabilized further by interheavy chaindisulfide bridges in the hinge region of the antibody. Specifically, inIgG4, the hinge region contains the amino acid sequence Cys-Pro-Ser-Cys(SEQ ID NO: 49) (in comparison to the stable IgG1 hinge region whichcontains the sequence Cys-Pro-Pro-Cys (SEQ ID NO: 50)) at amino acids226-230. This sequence difference of Serine at position 229 has beenlinked to the tendency of IgG4 to form novel intrachain disulfides inthe hinge region (Van der Neut Kolfschoten, M. et al., 2007, Science317:1554-1557 and Labrijn, A. F. et al, 2011, Journal of immunol187:3238-3246).

In another embodiment, the use of glutathione and glutathione disulfidecan be used in the production of bi-specific antibodies from twodistinct full antibodies. For example, the full antibodies, each whichrecognize different antigens, are incubated with reducing glutathione toseparate the antibodies into heavy-light chain heterodimers, ormolecules. The heavy-light chain heterodimers may be mixed with oxidizedglutathione (GSSG) which allows reassembly and reoxidation to formhighly pure bi-specific antibodies.

Therefore, bi-specific antibodies of the present invention can becreated through introduction of the R409 residue in the CH3 domain andthe Cys-Pro-Ser-Cys (SEQ ID NO: 49) sequence in the hinge region ofantibodies that recognize CCR4 or a second antigen, so that theheavy-light chain dimers exchange to produce an antibody molecule withone heavy-light chain dimer recognizing CCR4 and the second heavy-lightchain dimer recognizing a second antigen, wherein the second antigen isany antigen disclosed herein. Heavy-light chain heterodimer exchange canalso be enhanced with addition of a reducing agent, such as reducedglutathione, to promote the exchange. Known IgG4 molecules may also bealtered such that the heavy and light chains recognize CCR4 or a secondantigen, as disclosed herein. Use of this method for constructing thebi-specific antibodies of the present invention may be beneficial due tothe intrinsic characteristic of IgG4 molecules wherein the Fc regiondiffers from other IgG subtypes in that it interacts poorly witheffector systems of the immune response, such as complement and Fcreceptors expressed by certain white blood cells. This specific propertymakes these IgG4-based bi-specific antibodies attractive for therapeuticapplications, in which the antibody is required to bind the target(s)and functionally alter the signaling pathways associated with thetarget(s), however not trigger effector activities.

In some embodiments, mutations are introduced to the constant regions ofthe bsAb such that the antibody dependent cell-mediated cytotoxicity(ADCC) activity of the bsAb is altered. For example, the mutation is anLALA mutation in the CH2 domain, wherein the leucines at positions 234and 235 of the Fc region is mutated to alanine, and abrogates binding byspecific Fc receptors. In one aspect, the bsAb contains mutations on onescFv molecule of the heterodimeric bsAb, which reduces the ADCCactivity. In another aspect, the bsAb contains mutations on both chainsof the heterodimeric bsAb, which completely ablates the ADCC activity.For example, the mutations introduced one or both scFv molecules of thebsAb are LALA mutations in the CH2 domain. These bsAbs with variableADCC activity can be optimized such that the bsAbs exhibits maximalselective killing towards cells that express one antigen that isrecognized by the bsAb, however exhibits minimal killing towards thesecond antigen that is recognized by the bsAb.

The present invention provides for bi-specific antibodies that recognizeCCR and a second antigen. In one embodiment, the second antigen isPD-L1. In another embodiment, the second antigen is CAIX. In otherembodiments the second antigen is CA-IX, PD-L1, IL21, IL21R, HVEM,CD160, TIM3, GITR, LAG3 or GALS.

The bi-specific antibodies disclosed herein may be useful in treatmentof diseases or medical conditions, for example, cancer. The cancer is,for example, a solid cancer, such as renal cell carcinoma, breast canceror prostate cancer. In other embodiments, the cancer is a cancer inwhich CAIX, PD-L1 or HVEM is overexpressed when compared to tissue or asubject that does not have cancer. The bi-specific antibodies of thepresent invention may be used to treat, prevent, or alleviate a symptomof the cancer.

The bi-specific antibodies of the present invention may be used toincrease T cell proliferation, in which the T cell is a regulatory Tcell. The bi-specific antibodies of the present invention may beparticularly useful for promoting or augmenting a T cell response, suchas an antigen-specific T cell response. The bi-specific antibodies ofthe present invention can also be useful for reversing regulatory Tcell-mediated suppression of effector T cell proliferation.

Fusion Proteins

The invention provides a fusion protein containing a CCR4-IgG4 antibodydisclosed herein, or a functional fragment thereof, operably linked to asecond protein. The second protein can be, for example, a cytokine or agrowth factor. In particularly preferred embodiments, the cytokine isIL-2 or TGF-beta. In some other embodiments, the second protein may be atherapeutic agent, such as a toxin, or a detectable moiety, such as afluorescent protein for detection. In some embodiments, the CCR4-IgG4antibodies of the present invention may be operably linked to more thanone additional protein or peptide, for example 2, 3, 4, 5, 6, 7, 8, 9,or 10 additional proteins or peptide sequences.

In some embodiments, the CCR4-IgG4 antibody disclosed herein, orfunctional fragment thereof, is joined directly to the second protein.In other embodiments, the CCR4-IgG4 antibody, or functional fragmentthereof, is joined to the second protein via a linker, such as aflexible polypeptide chain. The linker can be any suitable linker of anylength, but can be at least 1, 2, 3, 4, 5, 10, 15, 20, 25, or 30 aminoacids in length. In one embodiment, the linker is an amino acid sequencethat is naturally present in immunoglobulin molecules of the host, suchthat the presence of the linker would not result in an immune responseagainst the linker sequence by the mammal. Fusion proteins of thepresent invention that include more than one additional protein to theCCR4-IgG4 antibody may have multiple linker sequences that join eachadditional protein or peptide sequence.

The fusion proteins of the present invention may be constructed byrecombinant methods known to the skilled artisan. For example, anexpression vector containing the nucleic acid sequence encoding aCCR4-IgG4 antibody of the present invention can be operably linked tothe nucleic acid sequence encoding the second protein and can beintroduced to an expression system to translate and produce the fusionprotein. Alternatively, one skilled in the art could readily utilize denovo protein synthesis techniques to produce the fusion proteinsdescribed herein.

Methods of Treatment

The invention provides for both prophylactic and therapeutic methods oftreating a subject at risk of (or susceptible to) a cancer, or othercell proliferation-related diseases or disorders. Such diseases ordisorders include but are not limited to, e.g., those diseases ordisorders associated with aberrant expression of CCR4. For example, themethods are used to treat, prevent or alleviate a symptom of ahematologic cancer such cutaneous T-cell Lymphoma (CTCL), mycosisfungoides (MF), primary cutaneous anaplastic large cell Lymphoma(cutaneous ALCL), Sezary syndrome, or adult T cell Leukemia/Lymphoma(ATLL). Alternatively, the methods are used to treat, prevent oralleviate a symptom of a solid tumor such as renal cell carcinoma,breast cancer, lung cancer, ovarian cancer, prostate cancer, coloncancer, cervical cancer, brain cancer, liver cancer, pancreatic canceror stomach cancer. In other embodiments, the antibodies of the presentinvention, such as bi-specific antibodies of the present invention, canbe used for the treatment of cancers that are characterized by CAIX,PD-L1, HVEM-overexpressing tumors. For example, the bi-specific antibodythat recognizes CAIX and CCR4 may be used for treatment of a cancer withtumors that overexpress CAIX. For example, the bi-specific antibody thatrecognizes PD-L1 and CCR4 may be used for treatment of a cancer withtumors that overexpress PD-L1. For example, the bi-specific antibodythat recognizes HVEM and CCR4 may be used for treatment of a cancer withtumors that overexpress HVEM.

Accordingly, in one aspect, the invention provides methods forpreventing, treating or alleviating a symptom cancer or a cellproliferative disease or disorder in a subject by administering to thesubject a monoclonal antibody or scFv antibody of the invention. Forexample, a CCR4-IgG4 antibody may be administered in therapeuticallyeffective amounts.

Subjects at risk for cancer or cell proliferation-related diseases ordisorders include patients who have a family history of cancer or asubject exposed to a known or suspected cancer-causing agent.Administration of a prophylactic agent can occur prior to themanifestation of cancer such that the disease is prevented or,alternatively, delayed in its progression.

In another aspect, tumor cell growth is inhibited or suppressor T-cellactivity is decreased by contacting a cell with a CCR4 antibody of theinvention. The cell is any cell that expresses CCR4. For example thecell is T-cell.

Also included in the invention are methods of increasing or enhancing animmune response to an antigen. An immune response is increased orenhanced by administering to the subject a monoclonal antibody or scFvantibody of the invention. The antigen is a viral (e.g. HIV), bacterial,fungal or tumor antigen. The immune response is a natural immuneresponse. By natural immune response is meant an immune response that isa result of an infection. The infection is a chronic infection.

Alternatively, the immune response is a response induced due to avaccination. Accordingly, in another aspect the invention provides amethod of increasing vaccine efficiency by administering to the subjecta monoclonal antibody or scFv antibody of the invention and a vaccine.The antibody and the vaccine are administered sequentially orconcurrently. The vaccine is a tumor vaccine a bacterial vaccine or aviral vaccine.

The immune response is augmented for example by augmenting antigenspecific T effector function.

Combinatory Methods

The invention provides treating cancer in a patient by administering twoantibodies that bind to the same epitope of the CCR4 protein or,alternatively, two different epitopes of the CCR4 protein. Also, thecancer is treated by administering a first antibody that binds to CCR4and a second antibody that binds to a protein other than CCR4.

Additionally, the invention provides administration of an antibody thatbinds to the CCR4 protein and an anti-neoplastic agent, such a smallmolecule, a growth factor, a cytokine or other therapeutics includingbiomolecules such as peptides, peptidomimetics, peptoids,polynucleotides, lipid-derived mediators, small biogenic amines,hormones, neuropeptides, and proteases. Small molecules include, but arenot limited to, inorganic molecules and small organic molecules.Suitable growth factors or cytokines include an IL-2, GM-CSF, IL-12, andTNF-alpha. Small molecule libraries are known in the art. (See, Lam,Anticancer Drug Des., 12:145, 1997.)

The invention will be further described in the following examples, whichdo not limit the scope of the invention described in the claims.

EXAMPLES Example 1: General Methods

Antibodies and Flow Cytometry Analysis

IgG and scFv-Fcs format of mAb2-3 and KM2760 were constructed by cloningthe single-chain variable region (scFv) into pcDNA3.1-Hinge vector inframe with human IgG1 Fc region and by cloning heavy-chain variableregion (VH) and light-chain variable region (VL) into TCAE5.3 vector.For IgG4 cloning, the cDNA sequence of immunoglobulin heavy constantgamma 4 was from GenBank: BC111019.1 and used to replace the IgG1 Fcregion in TCAE5.3 constructing mAb2-3 IgG4. Similarly, stabilized formof IgG4 (see additional data) have also been constructed (see new ref).Antibodies were produced in 293T or 293F cells and purified byproteinA-Sepharose (Amersham) affinity chromatography.

Chemotaxis

Treg cells were placed in Transwell-migration wells (5 μM pore; Corning)with or without mAb2-3 IgG1 or mAb2-3 IgG4 for 3 h at 37° C., andmigrated cells harvested from the bottom chamber containing conditionedmedium from either IGROV-1, OVCAR-5 or OVCAR-8 cells. Percentages ofmigrated cells were calculated by dividing the number of transmigratedTreg by the number of input cells. Human CD4+ T cells were isolated byCD4+ T cell isolation kit (Miltenyi Biotech) and placed inTranswell-migration assays with mAb2-3 IgG1 or mAb2-3 IgG4 for 3 h at37° C., and migrated cells (CD4+CD25high) were enumerated as above inresponse to conditioned medium from either IGROV-1, OVCAR-5 or OVCAR-8cells. Percentages of migrated cells were calculated by dividing thenumber of transmigrated CD4+CD25high cells by the number of input cellswith comparable CD4+ and CD25+ levels.

Antibody-Dependent Cell Cytotoxicity Assay

ADCC assays were performed using the LDH release assay method. Briefly,SCID/Beige mouse neutrophils, human PBMCs, or purified human NK cellsand neutrophils were used as effector cells and Mac-1, Cf2Th-CCR4, orCf2Th cells were used as target cells. Target cells were plated at adensity of 1×10⁴ cells/well into 96-well plates and then antibodies wereadded at an appropriate concentration. After one-hour incubation, fresheffector cells were added to achieve an appropriate E/T ratio. Afterincubation at 37° C. (PBMCs for 4 hours, NK cells for 16 hours andneutrophils for 6 hours), the supernatants from each well were recoveredby centrifugation at 300×g for 5 min. The supernatant were measuredusing a nonradioactive cytotoxicity assay kit (Promega, Wis.). Theabsorbance at 490 nm of the plates was determined using an ELISA reader.For 51Cr release assay, 1×106 Mac-1 cells were labeled with 100 μCi (3.7MBq) of Na51Cr (Amersham International) for 1 h at 37° C., washedextensively, and used as targets. 51Cr-labeled target cells (5000 perwell) were seeded into 96-well plates. Experiments were conducted intriplicates at various PBMC (effector) to Mac-1 (target) ratios of12.5:1, 25:1, and 50:1, incubated at 37° C. for 4 hours, and then therelease of 51Cr into supernatants was determined. The cytotoxicity wascalculated by the following formula:

% Cytotoxicity=100×(E−SE−ST)/(M−ST) where E is the experimental releaseof the LDH form the target cells incubated with effector cells andantibody, SE the spontaneous release of the LDH from the effector cells,ST the spontaneous release of the LDH from the target cells and M is themaximum release of the LDH from the target cells incubated with 10%triton-X.

Regulatory T Cell Suppression Assay

CD4+CD25high and CD4+CD25− T cells were sorted by FACSCanto II flowcytometer using anti-CD4 and anti-CD25 antibodies (Biolegend).CD4+CD25-Teffs (2500 cells) were cultured with or without CD4+CD25highTregs (2500 or 1250 cells) in round-bottom 96-well plates coated withbound anti-CD3 (0.05 μg/ml) and soluble anti-CD28 (1 μg/ml) antibodies(Biolegend, CA). 25,000 irradiated (300 rad) CD3-depleted PBMCs with orwithout c1567IgG were added into the cocultured wells. Proliferation ofT cells was measured by incorporation of 3H-thymidine on day 5 using ascintillation counter. The percent proliferation of Teffs in Tregscocultures in all analyses was normalized to the proliferation of Teffsin mono-Teffs culture; the proliferation of mono-Teffs culture wasconsidered 100% for this normalization. For activation, plates werecoated with anti-CD3 at 37° C. for 2 hours and washed twice with PBS.

CCR4+ CTCL Tumor-Bearing Mouse Model

Human cancer xenografts were established in SCID/Beige mice (CharlesRiver). Cells were injected subcutaneously into the dorsolateral flankin 6-week mice. After 24 hours of injection, mice were randomly assignedto different treatment groups and treated with 3 mg/kg of mAb2-3 IgG1 ormAb2-3 IgG4, and mouse IgG4 (twice a week for three weeks) or 5 mg/kg ofcontrol-scFv-Fc, c1567-scFv-Fc, h1567-scFv-Fc, and equivalent volumes ofsaline by i.p. injection (twice a week for four weeks). Mouse bodyweight and tumor size were measured and monitored twice a week usingdigital caliper or Xenogen imaging. The tumor volumes were calculatedusing the equation, length×(width)2×0.52. Animal care was carried out inaccordance with the guidelines of Animal Care and Use Committee ofDana-Farber Cancer Institute.

Statistical Analyses

Data was analyzed using two-sided unpaired Student's t-test. Weconsidered a P value below 0.05 as significant for all analyses. Allvalues are represented as mean±standard deviation (S.D)

Statistical analyses were performed using 2-way ANOVA with Bonferronipost hoc tests and unpaired 2-tailed t-tests using GraphPad Prism 5(GraphPad Software, Inc., La Jolla, Calif.). P values less than 0.05were considered statistically significant.

In Vitro Antibody-Dependent Cell Cytotoxicity Assay

ADCC was performed using the lactate dehydrogenase (LDH) release assaymethod, according to the CytoTox96 non-radioactive cytotoxicity assayprocedure specified by the manufacturer (Promega, Madison, Wis.). Mouseneutrophils purified from SCID-BEIGE mouse or purified human NK cellsfrom PBMC was used as effector cells and CCR4+ Mac1 tumor cells wereused as target cells. Briefly, purified SCID-BEIGE mouse neutrophils orNK cells were plated at a density of 1×10⁴ cells per well in around-bottom 96-well plate in the presence of h1567 or 11A minibodies.After 1-hour of incubation, freshly prepared effector cells were addedat an effector-target cell ratio (E:T) of 80:1 (mouse neutrophils) or2:1 (human NK cells). After 2 h incubation at 37° C., supernatants ofeach well were recovered by centrifugation at 300×g for 5 min. LDHactivity in the supernatant was determined by measuring absorbance at awavelength of 490 nm. The cytotoxicity (%) was calculated according tothe following formula:

% Cytotoxicity=100×(E−SE−ST)/(M−ST) where E is the LDH release byeffector-target coculture, SE the spontaneous release of the LDH fromthe effector cells, ST the spontaneous release of the LDH from thetarget cells and M the maximum release of the LDH from the target cellsincubated with lysis solution (10% Triton-X). All measurements were donein triplicate.

Example 2: In Vitro Treg Chemoattraction Mediated by CLL2 is Inhibitedby mAB2-3 IgG4 in a Dose Dependent Manner

The ovarian cancer cell lines IGROV-1, OVCAR-5, and OVCAR-8 were assayedfor expression of the CCR4 ligand, CCL22 by FACS. All ovarian cancercell lines had appreciable levels of CCL22 expression, with theexception of OVCAR-8. Cell culture supernatant from each of the threecancer cell lines was used for chemoattraction assays. Specifically,transwell assays were performed to assess the migration of CD4+/CD25+Treg cells to the bottom chamber of the transwell which containedsupernatant from one of the three ovarian cancer cell lines. Tregsincubated in transwells that had ovarian cancer cell conditioned mediumresulted in 100% migration of the cells to the bottom chamber. Incontrast, transwell assays in which the bottom chamber contained freshculture medium resulted in less than 40% migration of the Treg to thebottom chamber. Addition of either, mAb2-3 IgG1, or mAb2-3 IgG4 resultedin a statistically significant reduction in the migration of Tregs tothe bottom chamber when the bottom chamber contained supernatant fromIGROV-1 cells or OVCAR-5 cells. (See FIG. 1A-B).

The effect of mAb2-3 IgG4 dosage on the ability to inhibit the migrationof human lymphocytes to the bottom chamber of a transwell containingCCL22 rich medium demonstrates that 0.8 μg/ml results in a decrease inthe migration of human lymphocytes by greater than 50% to the bottomchamber, whereas addition of 20 μg/ml results in a near completeinhibition of transwell migration. (See FIG. 3B).

Example 3: In Vivo Treg Chemoattraction Mediated by CLL2 is Inhibited bymaB2-3 IgG4

INGROV-1 cells were injected subcutaneously into the dorsolateral flankof immunocompromised mice, followed by a period of growth. Micesubsequently received a tail vein injection of Treg cells labeled withluciferase (CD4+/CD25+/CD127dim/−). These mice were then treated witheither 3 mg/kg mAb2-3 IgG1, 3 mg/kg mAb2-3 IgG4, or equal volume of PBSas a control, in order to assess the migration of the labeled Tregs tothe site of the tumor. In vivo bioluminescence assays demonstrated thatthe migration of Tregs to the tumor area was lessened following theadministration of either mAb2-3 IgG1 or mAb2-3 IgG4. (See FIG. 2 ).

Example 4: mAb2-3 IgG4 does not Result in Lymphocyte Depletion

Human peripheral blood mononuclear cells (PBMCs) were injected via tailvein into immunocompromised mice and allowed to incorporate into thecirculation. Mice were subsequently administered either mAb2-3 IgG1 ormAb2-3 IgG4, followed by a 24 hour rest period prior to analysis of thePBMCs. Blood was collected and the amounts of human derived Tregs wereassessed by FACS analysis. The data demonstrate that following theadministration of mAb2-3 IgG1, there was a significant reduction of theamount of Tregs compared to those found after administration of the PBScontrol. In contrast, following administration of the mAb2-3 IgG4antibody, there was no statistically significant difference found in theamount of Tregs when comparing the amounts found followingadministration of PBS or a control IgG. These data demonstrate thatmAb2-3 IgG4 does not deplete the amount of total Tregs in thecirculation, whereas mAb2-3 IgG1 does result in Treg depletion.

Characterization of other T-cell populations within the human derivedPBMCs following administration revealed that there was a greater amountof T-effector cells and T-naïve cells following administration of mAb2-3IgG4 when compared to the amounts of these cells followingadministration of mAb2-3 IgG1. (See FIG. 3 ).

Collectively these data, and the data presented in Example 3, show thatmAb2-3 IgG4 modulates regulatory T-cell recruitment without inducing Tlymphocyte depletion.

Example 5: Tumor Primed T-Cells Mediate IGROV-1 Cell Death and SecreteIFN-γ Differentially in the Presence of Tregs Cells

In order to assess the dynamics of IFN-γ secretion by tumor-primedT-cells, INGROV-1 tumor primed T-cells were incubated with eithertumor-pulsed dendritic cells (DCs) or with unpulsed DCs, followed bymeasurement of IFN-γ levels in the medium. IGROV-1 tumor primed T-cellsmaintained high levels of IFN-γ secretion following incubation withtumor-pulsed dendritic cells (DCs) or with co-incubation with CD3/CD28beads. In contrast, unpulsed DCs co-incubated with tumor primed T-cells,resulted in less IFN-γ secretion by the tumor primed T-cells. Additionalexperiments aimed to assess the influence of Treg cells on the secretionof IFN-γ by the tumor primed T-cells. These data reveal a reduction inthe IFN-γ levels following incubation with either autologous orallogeneic Treg cells. (See FIG. 4B-C).

Additional in vitro studies aimed to assess the amount of IGROV-1 celldeath following incubation with tumor-primed T-cells in the presence ofeither autologous or allogeneic Treg cells. The data indicate areduction in the amount of IGROV-1 cell death following incubation withtumor-primed T-cells and either autologous or allogenic Treg cells, whencompared with tumor-primed T-cells incubated in the absence of eitherTreg population. (See FIG. 4D).

Example 6: In Vivo Reduction of Tumor Size Following Administration ofmAb2-3 IgG4

Immunocompromised mice received 5×10⁶ IGROV-1 tumor cells, and tumorswere allowed to grow until these reached an average of 50 mm³.Subsequently, 1×10⁷ IGROV-1 primed T-cells, 1×10⁶ Treg cells, and either1 mg/kg mAb2-3 IgG1, 1 mg/kg mAb2-3 IgG4, control IgG4 or PBS wereinjected via the tail vein. The antibodies were administered twice perweek for a total of 35 days. The data from these experiments show thataddition of either mAb2-3 IgG1 or mAb2-3 IgG4 resulted in a significantdecrease in the size of the tumor compared to control IgG4 and PBS. (SeeFIG. 5A-B).

Example 7: Anti-CCR4 Antibodies on Proliferation of Teffs and theAbrogation of the Suppressive Function of Tregs

Human PBMCs or CD4+ T cells were incubated with Tregs at 10:1 ratio with20 μg/ml control IgG1, mAb2-3 IgG1, or mAb2-3 IgG4. The proliferation ofhuman PBMCs and the transduced signals from CD4+ T cells were furtherdetected by flow cytometry, western blot and staining. These data showedthat the addition of either mAb2-3 IgG1 or mAb2-3 IgG4 resulted in theinhibition of Treg activity on suppression of human PBMCs, reduced theexpression of SA-β-gal and p-P38, and restored the expression of CD27and CD28, which provide co-stimulatory signals and are required forlymphocyte activation.

REFERENCES

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Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

What is claimed is:
 1. A nucleic acid encoding an isolated humanizedmonoclonal antibody that binds to the human CC chemokine receptor 4(CCR4) and has an IgG4 heavy chain constant region, wherein the antibodycomprises: a heavy chain with three CDRs comprising a CDR1 comprisingamino acid sequence SEQ ID NO: 9, a CDR 2 comprising amino acid sequenceSEQ ID NO:11, and a CDR 3 comprising amino acid sequence SEQ ID NO:13;and a light chain with three CDRs comprising a CDR1 comprising aminoacid sequence SEQ ID NO:10, a CDR 2 comprising amino acid sequence SEQID NO:12, and a CDR 3 comprising amino acid sequence SEQ ID NO:14; aheavy chain with three CDRs comprising a CDR1 comprising amino acidsequence SEQ ID NO: 33, a CDR 2 comprising amino acid sequence SEQ IDNO: 11, and a CDR 3 comprising amino acid sequence SEQ ID NO:35; and alight chain with three CDRs comprising a CDR1 comprising amino acidsequence SEQ ID NO: 10, a CDR 2 comprising amino acid sequence SEQ IDNO: 12, and a CDR 3 comprising amino acid sequence SEQ ID NO:40; a heavychain with three CDRs comprising a CDR1 comprising amino acid sequenceSEQ ID NO:33, a CDR 2 comprising amino acid sequence SEQ ID NO:11, and aCDR 3 comprising amino acid sequence SEQ ID NO:36; and a light chainwith three CDRs comprising a CDR1 comprising amino acid sequence SEQ IDNO:10, a CDR 2 comprising amino acid sequence SEQ ID NO:12, and a CDR 3comprising amino acid sequence SEQ ID NO:41; or a heavy chain with threeCDRs comprising a CDR1 comprising amino acid sequence SEQ ID NO: 31, aCDR 2 comprising amino acid sequence SEQ ID NO:11, and a CDR 3comprising amino acid sequence SEQ ID NO:13; and a light chain withthree CDRs comprising a CDR1 comprising amino acid sequence SEQ IDNO:10, a CDR 2 comprising amino acid sequence SEQ ID NO:12, and a CDR 3comprising amino acid sequence SEQ ID NO:38; and a heavy chain constantregion comprising the amino acid sequence of SEQ ID NO: 6 or SEQ ID: 8,and wherein the antibody modulates regulatory T cell recruitment withoutinducing T lymphocyte depletion.
 2. The nucleic acid of claim 1, whereinthe isolated humanized monoclonal antibody comprises a variable heavychain region (V_(H)) comprising the amino acid sequence of SEQ ID NO: 2and a variable light chain region (V_(L)) comprising the amino acidsequence of SEQ ID NO: 4; a variable heavy chain region (V_(H))comprising the amino acid sequence of SEQ ID NO: 20 and a variable lightchain region (V_(L)) comprising the amino acid sequence of SEQ ID NO:22; a variable heavy chain region (V_(H)) comprising the amino acidsequence of SEQ ID NO: 24 and a variable light chain region (V_(L))comprising the amino acid sequence of SEQ ID NO: 25; or a variable heavychain region (V_(H)) comprising the amino acid sequence of SEQ ID NO: 44and a variable light chain region (V_(L)) comprising the amino acidsequence of SEQ ID NO: 46; and the heavy chain constant region comprisesSEQ ID NO: 6 or SEQ ID:
 8. 3. The nucleic acid of claim 1, wherein saidantibody has a binding affinity of about 1.5 nM⁻¹ or less.
 4. Thenucleic acid according to claim 1, wherein the antibody is linked to atherapeutic agent.
 5. The nucleic acid of claim 4, wherein saidtherapeutic agent is a toxin, a radiolabel, a siRNA, a small molecule,or a cytokine.
 6. The nucleic acid of claim 5, wherein said cytokine isIL-2 or TGF-beta.
 7. The nucleic acid of claim 1, wherein the antibodycomprises a bi-specific antibody comprising the isolated humanizedmonoclonal antibody of claim 1 or 2 that binds to the human CC chemokinereceptor 4 (CCR4) and an antibody that immunospecifically binds to asecond antigen.
 8. The nucleic acid of claim 7, wherein the secondantigen is a tumor associated antigen or a T-cell function modulatingmolecule.
 9. The nucleic acid of claim 8, wherein the tumor associatedantigen is CA-IX, ErbB2 or HVEM.
 10. The nucleic acid of claim 8,wherein the T-cell function modulating molecule is PD-L1, GITR, IL21,IL21R, CD160, TIM3, LAG3 or GALS.
 11. A cell comprising the nucleic acidof claim
 1. 12. A method of inhibiting the migration of regulatoryT-cells (Tregs) in a subject by administering to said subject thenucleic acid of claim
 1. 13. A method of claim 12, wherein lymphocytesare not depleted.
 14. A method of claim 12, wherein effector T-cells arenot depleted.
 15. A method of claim 12, wherein Tregs are not depleted.16. A method of augmenting an immune response to an antigen in a subjectcomprising administering to the subject the nucleic acid of claim
 1. 17.The method of claim 16, wherein said antigen is a viral antigen, abacterial antigen or a tumor associated antigen.
 18. The method of claim16, wherein said administration of said nucleic acid causes an increasein antigen specific T-cell activity.
 19. The method of claim 16, whereinsaid administration of said nucleic acid causes an increase in T-cellproliferation.
 20. The method of claim 16, wherein effector T-cells areaugmented.
 21. A method of reversing regulatory T cell-mediatedsuppression of effector T cell proliferation comprising contacting a Tcell with the antibody encoded by the nucleic acid of claim
 1. 22. Amethod of treating or alleviating a symptom of cancer, comprisingadministering to a subject in need thereof a composition comprising thenucleic acid according to claim
 1. 23. The method of claim 22, whereinsaid cancer is a solid cancer or a hematologic cancer.
 24. The method ofclaim 23, wherein said hematologic cancer is cutaneous T-cell Lymphoma(CTCL), mycosis fungoides (MF), primary cutaneous anaplastic large cellLymphoma (cutaneous ALCL), Sezary syndrome, or adult T cellLeukemia/Lymphoma (ATLL).
 25. The method of claim 23, wherein the canceris a solid cancer or a cancer that overexpresses CA IX, PD-L1, or HVEM.26. The method of claim 23, were said solid cancer is renal cellcarcinoma, breast cancer, lung cancer, ovarian cancer, prostate cancer,colon cancer, cervical cancer, brain cancer, liver cancer, pancreaticcancer, kidney or stomach cancer.
 27. A vector comprising the nucleicacid of claim
 1. 28. An isolated cell comprising the vector of claim 27.29. The nucleic acid of claim 1, wherein the nucleic acid comprises SEQID NO: 1 and SEQ ID NO: 3; SEQ ID NO: 19 and SEQ ID NO: 21; SEQ ID NO:23 and SEQ ID NO: 25; or SEQ ID NO: 43 and SEQ ID NO: 45; and whereinthe nucleic acid further comprises SEQ ID NO: 5 or SEQ ID NO:
 7. 30. Anucleic acid comprising the nucleic acid sequence of SEQ ID NOs: 1 and3, SEQ ID NOs: 19 and 21, SEQ ID NOs: 23 and 25, or SEQ ID NOs: 43 and45, wherein the nucleic acid further comprises SEQ ID NO: 5 or SEQ IDNO:
 7. 31. A vector comprising the nucleic acid of claim
 30. 32. Anisolated cell comprising the vector of claim 31.